Treatment of platelet derived growth factor related disorders such as cancers

ABSTRACT

The present invention concerns compounds which can inhibit platelet derived growth factor receptor (PDGF-R) activity, preferably such compounds also inhibit the activity other members of the PDGF-R super family and are selective for members of the PDGF-R super family. The PDGF-R super family includes PDGF-R and PDGF-R related kinases Flt, and KDR. The featured compounds are active on cell cultures to reduce the activity of the PDGF-R and preferably one or more PDGF-R related kinases. An example of a featured compound, A10 (see FIG. 1a), and its ability to inhibit growth of tumor cells in vivo is described below. Using the present application as guide other compounds able to inhibit PDGF-R and preferably Flt and/or KDR can be obtained. Such compounds are preferably used to treat patients suffering from cell proliferative disorders characterized by inappropriate PDGF-R activity.

This application is a Continuation in Part of U.S. application Ser. No.08/179,570 filed Jan. 7, 1994 now U.S. Pat. No. 5,700,823.

FIELD OF INVENTION

The present invention relates to methods and compositions for treatingcell proliferative disorders characterized by inappropriate plateletderived growth factor receptor (PDGF-R) activity.

RELATED APPLICATION

The present application is a continuation-in-part of Hirth et al.,entitled "TREATMENT OF PLATELET DERIVED GROWTH FACTOR RELATED DISORDERSSUCH AS CANCERS" U.S. Ser. No. 08/179,570, filed Jan. 7, 1994, theentire contents of which including the drawings are hereby incorporatedby reference herein.

BACKGROUND OF THE INVENTION

Platelet derived growth factor receptor (PDGF-R) is a transmembranereceptor tyrosine kinase. Ligand binding to the receptor results indimerization of two receptors generally leading to intermolecularphosphorylation of each receptor, commonly referred to asautophosphorylation or transphosphorylation, and activation of thereceptor complex. PDGF, which is a ligand for PDGF-R, is a dimericprotein having two polypeptide chains joined by disulfide bonds. Eachpolypeptide is either an A chain polypeptide or a B chain polypeptide.Thus, PDGF can have either two A chains, two B chains, or an A and a Bchain.

The PDGF-R consists of two isozymes α and β. Both α and β-containingreceptors have been associated with mitogen activity, while only theβ-containing receptor has been associated with chemotaxis and actinreorganization (Heldin, C-H, EMBO Journal 11:4251-4259, 1992).

According to Plate et al., Laboratory Investigation 4:529-534, 1992:

PDGF is a potent growth factor for mesenchymal and neuroectodermalcells. Endothelial cells have been considered nonresponsive to PDGF, buta recent study has shown that PDGF may have a role in angiogenesisduring placenta development. In addition, it has been demonstrated, thatPDGFR-b is expressed in endothelial cells in inflammatory tissue andglial tumors. This suggests, that PDGF may play a role in vascularfunctions in pathological conditions. [Citations omitted.]

Heldin, supra, describes the relationship of PDGF and its receptor, anddiscusses the role of PDGF in cancer, noting that some cancers do notproduce PDGF and have central necroses. Heldin states:

The adverse effects of PDGF in certain diseases, as discussed above,make PDGF antagonists highly desirable. We and others have recentlytaken several approaches to develop such antagonists. Antibodies againstPDGF have proven to be useful for inhibiting both autocrine stimulationin SSV-transformed cells and the atherosclerotic process that occursafter de-endothelialization of the carotid arteries of rats. Moreover, asoluble form of the PDGF receptor has been shown to bind and inactivatePDGF, and could thus be potentially useful for inhibiting PDGF action invivo.

Another approach would be to design or find agents that compete in anantagonistic manner with PDGF for receptor binding. In order to identifypeptides that interfere with PDGF binding, we systematically screenedpeptides derived from the B-chain sequence. One peptide was found thatinhibited PDGF binding and autophosphorylation of α- as well asβ-receptors. However, the peptide also showed some cell toxicity andfurther development will be necessary before peptide antagonists becomeuseful for in vivo studies. Low molecular weight compounds thatinterfere with receptor binding have been described, e.g., suramin.However, suramin is not specific enough to be clinically useful as aPDGF antagonist. We recently found that another low molecular weightcompound, neomycin, at high concentrations inhibited the binding ofPDGF-BB to the α-receptor, but did not inhibit binding to theβ-receptor. This compound thus represents an antagonist thatdistinguishes between the two receptor types; however, its low potencymakes it unsuitable for use in vivo. Hopefully, the experiences withsuramin and neomycin will aid the future design of more potent andspecific PDGF receptor antagonists. The design of such antagonists wouldbe much facilitated by the elucidation of the three-dimensionalstructure of the PDGF-receptor complex.

PDGF antagonistic activity could also be achieved by inhibition of PDGFreceptor dimerization. We hypothesized that monomeric PDGF might fail toinduce receptor dimerization and might thus have antagonistic activity.Since reduction of PDGF results in loss of receptor binding, weattempted to identify the interchain disulfide bonds in order to mutatethese residues and hereby prevent dimerization of the ligand. Thisturned out to be quite difficult due to the high density of cysteineresidues in PDGF. The approach that finally succeeded involved partialreduction of the PDGF molecule using a concentration of dithiothreitolthat reduced only the interchain disulfide bonds, and left theintrachain bonds unaffected. By this procedure the second and fourthcysteine residues from the N-terminus were found to form the twointerchain bonds in PDGF. Analysis of a PDGF B-chain mutant in whichthese two cysteine residues had been mutated to serine residues revealedthat it retained receptor binding activity. Is it a receptor antagonist?The answer is no, in fact, the monomeric PDGF induced both receptordimerization and autophosphorylation. This result may indicate thatPDGF-induced receptor dimerization is not only a matter of forming abridge between two receptor molecules: the dimerization may also involvea ligand-induced conformational change of the extracellular domains ofthe receptors which promotes receptor-receptor interactions. Onepossible way of achieving an antagonistic effect, which we are currentlyexploring, is to combine a wild-type PDGF chain with a mutated chainthat does not bind to PDGF receptors but can actively preventdimerization of receptors. [Citations omitted.]

Spada A. P., et al., entitled "Bis Mono- and Bicyclic Aryl andHeteroaryl Compounds Which Inhibit EGF and/or PDGF Receptor TyrosineKinase," PCT/US92/03736, mentions the use of certain bis mono andbicylic aryl compounds. According to Spada:

In accordance with the present invention, there is provided a method ofinhibiting abnormal cell proliferation in a patient suffering from adisorder characterized by such proliferation comprising theadministration to a patient of an EGF and/or PDGF receptor inhibitingeffective amount of a bis mono- and/or bicyclic aryl and/or heteroarylcompound exhibiting protein tyrosine kinase inhibition activity whereineach aryl and/or heteroaryl group is a ring system containing 0-4 heteroatoms, said compound being optionally substituted or polysubstituted.

SUMMARY OF THE INVENTION

The present invention concerns compounds which can inhibit plateletderived growth factor receptor (PDGF-R) activity, preferably suchcompounds also inhibit the activity other members of the PDGF-R superfamily and are selective for members of the PDGF-R super family. ThePDGF-R super family includes PDGF-R and PDGF-R related kinases Flt, andKDR. The featured compounds are active on cell cultures to reduce theactivity of the PDGF-R and preferably one or more PDGF-R relatedkinases. An example of a featured compound, A10 (see FIG. 1a), and itsability to inhibit growth of tumor cells in vivo is described below.Using the present application as guide other compounds able to inhibitPDGF-R and preferably Flt and/or KDR can be obtained. Such compounds arepreferably used to treat patients suffering from cell proliferativedisorders characterized by inappropriate PDGF-R activity.

Unwanted cell proliferation can result from inappropriate PDGF-Ractivity occurring in different types of cells including cancer cells,cells surrounding a cancer cell (stromal cells), endothelial and smoothmuscle cells. For example, an increase in PDGF-R activity of endothelialcells surrounding cancer cells may lead to an increased vascularizationof the tumor, thereby facilitating growth of the cancer cells. Thus,inappropriate PDGF-R activity can contribute to a cell proliferativedisorder in different ways such as through increasing the production ofgrowth factors, causing aberrant growth of a cell, and increasingformation and spreading of blood vessels in solid tumors therebysupporting tumor growth.

Other members of the PDGF-R super family are also involved in supportingtumor growth. Member of the PDGF-R super family have a kinase domaincontaining at least 45% sequence similarity with the kinase domain of α-or β-PDGF-R. Vascular endothelial growth factor (VEGF) activates atleast tyrosine kinase receptors; Flk-1 or its human homologue KDR, andFlt-1. Both of these receptors are expressed on endothelial cell andappear to be important in angiogenesis. Plate et al., Nature359:845-848, 1992; Shweiki, et al., Nature 359:843-845, 1992; Millaueret al., Cell 72:835-846, 1993; Plate et al., Cancer Res., 53:5822-5827,1993; Waltenberger et al., Journal of Biological Chemistry43:26988-26995, 1994. Vascularization is essential for solid tumorgrowth and is thought to be regulated by tumor cell factors which havechemotactic and/or mitogenic effects of endothealial cells. PDGF-R, KDRand Flt-1, are all involved in blood vessel formation and spreadingfeeding solid tumors. By inhibiting both PDGF-R and one or more relatedtyrosine kinase activities both aberrant cell growth and the feeding ofsuch growth can be inhibited.

Many examples of compounds (see FIGS. 1a-k) belonging to the featuredgroups (see FIGS. 2a-j) are described. Those skilled in the art canobtain other compounds, to inhibit PDGF-R and preferably Flt and/or KDR,having equivalent or greater activity at these receptor tyrosine kinasesusing the present disclosure as a guide. For example, the assaysdescribed herein can be used to readily screen other compounds belongingto the featured groups (see, FIGS. 2a-j) for equivalent activity. Usingstandard assays, the site of action of any one of the compoundsdescribed below may be determined and other compounds active at the samesite determined.

The methods and compositions are designed to inhibit unwanted cellproliferation by altering the activity of the PDGF-R, and preferablyalso altering activity of Flt and/or KDR. Without being bound to anytheory, inhibition of unwanted cell proliferation may be brought aboutby altering the activity of the PDGF-R (e.g., by inhibiting tyrosinephosphorylation of PDGF-R, by inhibiting substrate or adaptor proteinbinding to the receptor, or by inhibiting other downstream signalingevents), thereby inhibiting the activity of the PDGF-R. However, unlessotherwise stated, the use of the claimed methods and compositions arenot limited to this particular theory.

Thus, a first aspect of the present invention features a method fortreating a patient inflicted with a cell proliferative disordercharacterized by inappropriate PDGF-R activity. The method involves thestep of administering to the patient a therapeutically effective amountof a composition comprising a compound illustrated in FIGS. 2a-j, or theactive product formed when any such compound is placed underphysiological conditions (i.e., the active structural entity of apro-drug described above). Administration of a particular compound isachieved by providing a particular compound to the patient or providinga prodrug of the compound to a patient which forms the particularcompound in vivo.

"Cell proliferative disorders" refer to disorders wherein unwanted cellproliferation of one or more subset of cells in a multicellular organismoccurs resulting in harm (e.g., discomfort or decreased life expectancy)to the multicellular organism. Cell proliferative disorders can occur indifferent types of animals and in humans. Cell proliferative disordersinclude cancers, blood vessel proliferative disorders, and fibroticdisorders.

"Inappropriate PDGF-R activity" refers to either 1) PDGF-R expression incells which normally do not express PDGF-R; 2) PDGF expression by cellswhich normally do not express PDGF; 3) increased PDGF-R expressionleading to unwanted cell proliferation; 4) increased PDGF expressionleading to unwanted cell proliferation; or 5) mutations leading toconstitutive activation of PDGF-R. The existence of inappropriate orabnormal PDGF and PDGF-R levels or activities is determined byprocedures well known in the art.

The compositions can be used to treat a cell proliferative disorder byadministering a therapeutically effective amount of the composition to apatient (i.e. a human or an animal having a cell proliferativedisorder). The compositions may also be used in in vitro studies of themechanism of action of the PDGF-R or PDGF itself.

A "therapeutically effective amount", in reference to the treatment of acancer refers to an amount sufficient to bring about one or more or thefollowing results: reduce the size of the cancer, inhibit the metastasisof the cancer, inhibit the growth of the cancer, stop the growth of thecancer, relieve discomfort due to the cancer, or prolong the life of apatient inflicted with the cancer.

A "therapeutically effective amount", in reference to the treatment of acell proliferative disorder other than a cancer refers to an amountsufficient to bring about one or more of the following results: inhibitthe growth of cells causing the disorder, relieve discomfort due to thedisorder, or prolong the life of a patient suffering from the disorder.

"Significant" inhibition of a receptor tyrosine kinase activity refersto an IC₅₀ of less than or equal to 75 μM using one or more of theassays described in the Examples infra. Preferably, the compound caninhibit PDGF-R activity with an IC₅₀ of less than or equal to 50 μM,more preferably less than or equal to 10 μM, more preferably less thanor equal to 1 μM. Lower IC₅₀ are preferred because the IC50 provides anindication as to the in vivo effectiveness of the compound. Otherfactors known in art, such as compound half-life, biodistribution, andtoxicity should also be considered for therapeutic uses. Such factorsmay enable a compound with a lower IC₅₀ to have greater in vivo efficacythan a compound having a higher IC₅₀.

Selective inhibition of the PDGF-R super family is achieved bysignificantly inhibiting PDGF-R activity, while having an insignificanteffect (i.e. an IC₅₀ for tyrosine phosphorylation greater than 100 μM onEGF-R. Preferably, at least one other member of the PDGF-R super family,is significantly inhibited.

Preferably, the compound is either A10, A11, A12, A13, B10, B11, B12,B13, B14, B15, B16, B17, B18, B19, C10, C11, C13, D11, D12, D13, D14,D15, D16, D17, D18, D20, E10, E11, E12, E13, E14, E15, E16, F10, F11,F12, G10, G11, G12, G13, G14, G15, G16, G17, C,18, G19, G20, G21, G22,G23, G24, G25, G27, G28, G29, G30, H12, I10, J11, P10, P12, P13, P14,P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25 or the active drugof such compounds, or pharmaceutically acceptable salts thereof. Thecompound is preferably used in a pharmaceutical composition formed bymixing one of the above compounds and a physiological acceptablecarrier.

A physiological acceptable carrier is a formulation to which thecompound can be added to dissolve or otherwise facilitate administrationof the compound. Examples of physiological acceptable carriers includewater, saline, physiologically buffered saline, cyclodextrins andPBTE:D5W. Hydrophobic compounds such as A10 are preferably administeredusing a carrier such as PBTE:D5W. An important factor in choosing anappropriate physiological acceptable carrier is choosing a carrier inwhich the compound remains active or the combination of the carrier andthe compound produces an active compound. The compound may also beadministered in a continuous fashion using a slow release formulation ora pump to maintain a constant or varying drug level in a patient.

Another aspect of the present invention features a method of treating apatient suffering from a cell proliferative disorder characterized byinappropriate PDGF-R activity using A10, A12, or B11. The methodinvolves administering to a patient a therapeutically effective amountof A10, A12, or B11.

Another aspect of the present describes a method of treating a patientsuffering from a cancer characterized by inappropriate PDGF-R activityusing combination therapy. Combination therapy is carried out using oneor more agent described herein along with standard anti-cancer agents.The method is carried out by administering to a cancer patient atherapeutically effective amount of a composition comprising an agentable to significantly inhibit PDGF-R activity and a cytotoxic agent.Preferably, the cytotoxic agent is VP-16 or cisplatin. More preferably,the cytotoxic agent is cisplatin and the cancer is lung cancer.

Another aspect of the present invention features a method for treating apatient having a cell proliferation disorder characterized byinappropriate PDGF-R activity using mutated PDGF-R, or nucleic acidencoding a mutated PDGF-R. "Mutated" PDGF-R refers to PDGF-R wherein oneor more amino acid is missing or altered. As illustrated below a nucleicacid encoding a mutated (i.e., a truncated) PDGF-R lacking a kinasedomain can inhibit tumor growth in vivo. Mutated PDGF-R can beadministered as a protein, or recombinant nucleic acid encoding theprotein and expressing the protein inside a cell.

In other aspects, the invention features novel compositions includingone of the featured compounds herein described and PBTE:D5W carrierwhere the featured compound is soluble in PBTE:D5W; and the novelcompounds B10, B12, C10, C11, E10, E11, E12, E13, E14, E15, E16, F10,F11, F12, G21, G22, H11, H12, H13, and H14.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-k illustrate the chemical structures of the preferredcompounds.

FIGS. 2a-j illustrate the generic chemical structure of groups 1-10respectively.

FIG. 3. NIH3T3 cells overexpressing the human PDGF-b (A) or the humanEGF (B) receptor were treated with A10 as indicated. The percentage ofcells in the S phase of the cell cycle was determined by flow cytometry.

FIG. 4. In two separate experiments, C6 cells (1×10⁵ cells in 4 μL) wereimplanted into the cerebrum of BALB/c, nu/nu mice. A10 was administeredIP in 100 μL PBTE:D5W at the indicated doses every day starting one daypost-implantation. V=vehicle control. n=8 to 12 (Expt #1), or 5 (Expt#2) animals per group. *P<0.00001; **P<0.002; ***P<0.02 compared tovehicle control.

FIG. 5. In two separate experiments, C6 cells (5×10⁴ cells in 20 μL[Expt #1] or 5 μL [Expt #2]) were implanted into the cerebrum of athymicrats. A10 was administered IP in 500 μL PBTE at the indicated dosesevery day starting one day post-implantation. V=vehicle control. n=7 to8 animals per group. *P=0.0002; **P=0.0003 compared to vehicle control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods and compositions for treatingcell proliferative disorders characterized by inappropriate PDGF-Ractivity. The present application demonstrates the ability of compoundsable to significantly inhibit activity of Flt-1 and/or PDGF-R, andprovides examples of such compounds useful for treating a proliferativedisease, such as cancer. The compounds described herein can be used intreatment of other proliferative diseases associated with inappropriateexpression of PDGF-R, for example, blood vessel proliferative disordersand fibrotic disorders characterized by inappropriate PDGF-R activity.Using the present disclosure as a guide, those in the art can readilydetermine which of the compounds described herein are useful to treat aparticular proliferative disease.

A single target site, the presence of inappropriate PDGF-R activity, fora large number of disorders out of the many proposed targets in the artalong with compounds able to inhibit PDGF-R activity and preferably Fltand/or KDR activity are described by the present application.Preferably, PDGF-R activity along with KDR and/or Flt activities areinhibited by a single compound such as A10. Combinations of compounds ortypes of treatments can also be used to target different PDGF-R relatedtyrosine kinases. Examples of such combinations include using a PDGF-Ractivity inhibitory compounds along with a KDR inhibitory compound, andusing and PDGF-R nucleic acid to inhibit production of PDGF-R along witha KDR inhibitory compound.

Compounds (also referred to herein as "drugs") useful in this inventionbelong to at least eight different groups. The preferred compounds ofthese groups, and in other as yet undefined groups, that have generallyexhibited significant inhibition of PDGF receptor activity are shown inFIGS. 1a-k. While generic formulae are presented, those in the art willrecognize that those compounds useful in the invention can be determinedby screening procedures described herein and known in the art.

The ability of A10, truncated versions of a PDGF-R and other compoundsto inhibit tumor growth in animals; illustrates the effectiveness andefficacy of these compounds. such animal studies support theeffectiveness of the compounds by demonstrating that the compounds canbe effective in animals despite various problems which are inherentlyassociated with using compounds in animals to treat a particularailment. The inherent problems include the animal being comprised of aheterogeneous cell population, various consideraogical considerationssuch as bioavailability of the compound, the half life of the compound,and clearance of the compound. These inherent problems often prevent acompound from exerting a physiological effect.

Examples are provided below illustrating the ability of variouscompounds to inhibit PDGF-R phosphorylation. Examples are also providedillustrating the ability of the compound termed A10 (see FIG. 1a) toinhibit cancers in vivo. Rather, using the present disclosure as a guideone skilled in the art can use the featured methods and compositions toobtain additional inhibitory compounds and to target other cellproliferative disorders characterized by an inappropriate PDGF-Ractivity.

I. PDGF-R Super Family

A. PDGF-R Activity

Ligand binding to the PDGF-R induces the formation of receptor dimersand allosteric changes that activate the intracellular kinase domains,and results in the transphosphorylation and/or autophosphorylation ofthe receptor on tyrosine residues. Receptor phosphorylation stimulates aphysical association of the activated receptor with target molecules.Some of the target molecules are in turn phosphorylated, which transmitsthe signal to the cytoplasm. Other target molecules are notphosphorylated, but assist in signal transmission by acting as dockingor adapter molecules for secondary signal transducer proteins. Thesecondary signal transducer molecules generated by activated receptorsresults in a signal cascade that regulates cell functions such as celldivision. (See, Fry M. J. et al., Protein Science 2:1785-1797, 1993)

Thus, an increase in PDGF-R activity is characterized by an increase inone or more of the activities which can occur upon PDGF-R ligandbinding: (1) phosphorylation or autophosphorylation of PDGF-R, (2)phosphorylation of a PDGF-R substrate (e.g., PL 3-kinase, RasGAP, PLCγ,see Fry supra), (3) activation of an adapter molecule, and (4) increasedcell division. These activities can be measured using techniquesdescribed below and known in the art. For example autophosphorylation ofPDGF-R can be measured as described in the examples below using ananti-phosphotyrosine antibody, and increased cell division can beperformed as described below by measuring ³ H-thymidine incorporationinto DNA. Preferably, the increase in PDGF-R activity is associated withan increased amount of phosphorylated PDGF-R and DNA synthesis.

B. PDGF-R Related Kinases

PDGF-R related kinases Flt-1 and KDR can be activated by VEGF. VEGF is amonodimeric glycoprotein with structural homology to PDGF. Fourdifferent splice variants of VEGF have been isolated. Rosenthal, et al.,Growth Factors, 4:53-59, 1990; Conn, et al., Proc. Natl. Acad. Sci.(USA), 87:1323-1327, 1990; Houck, et al., Mol. endocrinol., 5:1806-1814,1991; two are secreted forms and two remain cell-associated. VEGF hasbeen shown to be upregulated by hypoxia and acts specifically onendothelial cells. Plate et al., Nature, 359:845-848, 1992; Shweike, etal., Nature 359:843-845, 1992.

KDR activity is characterized by an increase in one or more of theactivities which can occur upon VEGF ligand binding: (1) phosphorylationor autophosphorylation of KDR, (2) phosphorylation of a KDR substrate,(3) activation of an adapter molecule, and (4) increased cell division.

Flt-1 activity is characterized by an increase in one or more of theactivities which can occur upon VEGF ligand binding: (1) phosphorylationor autophosphorylation of Flt-1, (2) phosphorylation of a Flt-1substrate, (3) activation of an adapter molecule, and (4) increased celldivision.

II. Featured Compounds

Compounds of groups 1 to 11 are shown in FIGS. 2a-k.

A. Group 1 compounds

Group 1 compounds have the following basic structure: ##STR1## where R₁,R₂, R'₂, R"₂, and R'"₂ are independently selected from the groupconsisting of hydrogen, halogen, trihalomethyl, and NO₂ ; preferably R₁and R₂ are independently CF₃, NO₂ or hydrogen, and R'₂, R"₂, and R"₂ arehydrogen; and

R₃ is selected from the group consisting of hydrogen, carboxy, alkoxy,or carbalkoxy; preferably hydrogen, carboxy, or methyl.

Examples of group 1 compounds are listed in Table I and shown in FIG 1a.

                  TABLE I                                                         ______________________________________                                        Compound   R.sub.1     R.sub.2                                                                              R.sub.3                                         ______________________________________                                        A10        CF.sub.3    H      H                                               A11        H           CF.sub.3                                                                             H                                               A12        CF.sub.3    H      Carboxy                                         A13        CF.sub.3    H      CH.sub.3                                        ______________________________________                                    

These compounds are believed to act as prodrugs in that the ring iscleaved in vivo to yield active metabolites.

B. Group 2 compounds

Group 2 compounds have the following basic structure: ##STR2## where R₄and R₅ are independently halogen, hydrogen, trihalomethyl, or NO₂ ;preferably R₄ is CF₃ and R₅ is H; R₆ is either aryl, alkyl, alkenyl, oralkynyl;

is R₆ is alkyl or one of the substituents of the compounds listed inTable II. Examples of group 2 compounds are listed in Table II and shownin FIG 1b.

                  TABLE II                                                        ______________________________________                                        Compound   R.sub.4    R.sub.5                                                                             R.sub.6                                           ______________________________________                                        B10        NO.sub.2   H     CH.sub.3                                          B11        CF.sub.3   H     CH.sub.3                                          B12        CF.sub.3   H     4-fluorophenyl                                    B13        CF.sub.3   H     cyclohexyl                                        B14        CF.sub.3   H     2,2,3,3-                                                                      tetramethylcyclo-                                                             propyl                                            B15        CF.sub.3   H     pentafluorophenyl                                 B16        CF.sub.3   H     3-phenoxy-phenyl                                  B17        CF.sub.3   H     benzyl                                            B18        CF.sub.3   H     2-methylpropyl                                    B19        CF.sub.3   H     diphenylmethyl                                    ______________________________________                                    

C. Group 3 Compounds

Group 3 compounds have the following structure: ##STR3## where R₇, R'₇,and R₈ are independently halogen, OH, hydrogen, alkoxy, SH, NH₂, orC(CH₃)₃, preferably R'₇, R₇, and R₈ is independently selected from H,OH, and C(CH₃)₃ ; more preferably, R₇ and R₈ are OH; R₉ is aryl orhydrogen, preferably hydrogen or phenyl. Examples of group 3 compoundsare listed in Table III and shown in FIG 1c.

                  TABLE III                                                       ______________________________________                                        Compound   R'.sub.7 R.sub.7  R.sub.8                                                                              R.sub.9                                   ______________________________________                                        C10        H        OH       OH     phenyl                                    C11        H        OH       OH     H                                         C13        C(CH.sub.3).sub.3                                                                      OH       C(CH.sub.3).sub.3                                                                    H                                         ______________________________________                                    

D. Group 4 Compounds

Group 4 compounds have the following chemical structure: ##STR4## whereR₁₀ is either ═S, ═O, SH, OH, or NH₂ ; and R₁₁ is SH, OH, NH₂, ═C(CN)₂or aryl, preferably NH₂, ═C(CN)₂, or dihydroxyl-phenyl; or R₁₀ and R₁₁taken together are aryl, preferably 3-amino-4-cyano-5-pyrazole or1-phenyl-3-amino-4-cyano-5-pyrazole; and R₁₂ is hydrogen, aryl, alkyl,alkenyl, or alkynyl, preferably hydrogen, --(CH₂)₂ CN₂ or --(CH₂)₂N(CH₃)₂.

Examples of group 4 compounds are listed in Table IV and shown in FIG.1d.

                  TABLE IV                                                        ______________________________________                                        Compound  R.sub.10   R.sub.11   R.sub.12                                      ______________________________________                                        D11       =S         NH.sub.2   H                                             D12       NH.sub.2   =C(CN).sub.2                                                                             H                                             D13       =O         NH.sub.2   H                                             D14       3-amino-4-cyano-5-pyrazoyl                                                                      H                                                 D15       3-amino-4-cyano-5-pyrazoyl                                                                      --(CH.sub.2).sub.2 N(CH.sub.3).sub.2              D16       =O         3,4-dihydroxyl-                                                                          --(CH.sub.2).sub.2 N(CH.sub.3).sub.2          D17       =O         3,4-dihydroxyl-                                                                          --(CH.sub.2).sub.2 CN.sub.2                                        phenyl                                                   D18       3-amino-4-cyano-5-pyrazoyl                                                                      --(CH.sub.2).sub.2 N(CH.sub.3).sub.2              D20       1-phenyl-3-amino-4-cyano-5-                                                                     H                                                           pyrazoyl                                                            ______________________________________                                    

E. Group 5 Compounds

Group 5 compounds have the following chemical structure: ##STR5## whereR₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈ are independently hydrogen, halogen,alkoxy, OH, amino, alkylamino, or SH; preferably hydrogen or OH.

Examples of group 5 compounds are listed in Table V and shown in FIG.1e.

                  TABLE V                                                         ______________________________________                                        Compound R.sub.13                                                                              R.sub.14                                                                             R.sub.15                                                                            R.sub.16                                                                            R.sub.17                                                                            R.sub.18                            ______________________________________                                        E10      OH      OH     H     H     H     N(CH.sub.3).sub.2                   E11      OH      OH     H     H     OH    H                                   E12      H       H      OH    H     OH    H                                   E13      OH      OH     H     OCH.sub.3                                                                           H     H                                   E14      OH      OH     H     OC.sub.2 H.sub.5                                                                    H     H                                   E15      OH      OH     H     H     NO.sub.2                                                                            H                                   E16      OH      H      H     H     NO.sub.2                                                                            H                                   ______________________________________                                    

F. Group 6 Compounds

Group 6 compounds have the following chemical structure: ##STR6## whereR₁₉ is aryl, alkyl, alkenyl or alkynyl preferably2-(3,4,-dihydroxyphenyl) ethenyl; R₂₀ is an alkyl preferablyethylenehydroxy; or R₁₉ and R₂₀ are together aryl preferably amorpholine ring having a ═CH--(mono or dihydroxy-phenyl) substituent.

Examples of group 6 compounds are set forth in Table VI and shown inFIG. 1f.

                  TABLE VI                                                        ______________________________________                                        Compound   R.sub.19     R.sub.20                                              ______________________________________                                        F10        (CH.sub.2).sub.2 OH                                                                        CH=CH-3,3-                                                                    dihydroxyphenyl                                       F11        2-C=CH--(3,4-dihydroxyphenyl)morpholino                            F12        2-C=CH--(3-hydroxyphenyl)morpholino                                ______________________________________                                    

G. Group 7 Compounds

Group 7 compounds have the following chemical structure: ##STR7## whereb is an optional pi bond, Y and Z are independently carbon or nitrogen;

R₂₁ and R₂₂, are independently hydrogen, halogen, OH, SH, NH₂, NO₂,alkyl, alkenyl, alkynyl, alkoxy, benzoyl, CODH, or carbalkoxy,preferably OH, NO₂, CH₃, methoxy, benzoyl, or COOH; or R₂₁ and R₂₂together form an aromatic ring to give an aryl, preferably phenyl;

R₂₃ is hydrogen, halogen, ═O, OH, SH, NH₂, alkoxy, COOH, aryl,preferably or a substituted or unsubstituted anilino, a substituted orunsubstituted phenyl, hydrogen, COOH, ═O, alkoxy, or methoxy, providedthat if R₂₃ is ═O b is present as a bond;

R₂₄ is H, or aryl, preferably a substituted or unsubstituted anilino,phenyl, or 2-thienyl; and

R₂₅ is hydrogen, halogen, ═S, or ═O, wherein if R₂₅ is ═O or ═S, b ispresent as a bond; provided that if b is no bond, the adjacent nitrogenoptionally has a substituent selected from the consisting of hydrogen,alkyl, alkyleneamino, alkyleneaminoalkly, and alkylenecyano.

Examples of group 7 compounds are set forth in Table VII and shown inFIG. 1g.

                  TABLRE VII                                                      ______________________________________                                        Com-                                                                          pound b       Y     Z   R.sub.21                                                                            R.sub.22                                                                            R.sub.23                                                                            R.sub.24                                                                            R.sub.25                      ______________________________________                                        G10   bond    N     C   H     H     H     phenyl                                                                              H                             G11   bond    C     C   OCH.sub.3                                                                           OCH.sub.3                                                                           OCH.sub.3                                                                           phenyl                                                                              Cl                            G12   bond    C     C   OCH.sub.3                                                                           OCH.sub.3                                                                           H     phenyl                                                                              Cl                            G13   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            H     phenyl                                                                              H                             G14   bond    N     C   OCH.sub.3                                                                           OCH.sub.3                                                                           H     phenyl                                                                              H                             G15   bond    N     C   H     H     H     2-    H                                                                       thienyl                             G16   hond    N     N   H     H     H     phenyl                                                                              H                             G17   bond    N     C   OH    OCH.sub.3                                                                           H     phenyl                                                                              H                             G18   no bond N     C   CH.sub.3                                                                            CH.sub.3                                                                            ═O                                                                              phenyl                                                                              H                             G19   bond    N     C   H     CH.sub.3                                                                            H     phenyl                                                                              H                             G20   bond    N     C   CH    OH    H     phenyl                                                                              H                             G21   bond    N     C   H     benzoyl                                                                             H     phenyl                                                                              H                             G22   bond    N     C   phenyl    H     phenyl                                                                              H                               G23   bond    N     C   H     NO.sub.2                                                                            H     phenyl                                                                              H                             G24   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            3,4-  phenyl                                                                              H                                                                 dyhyd-                                                                        roxy-                                                                         phenyl                                    G25   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            COOH  phenyl                                                                              H                             G27   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            H     NO.sub.2                                                                            H                             G28   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            H     3-    H                                                                       bromo-                                                                        phenyl                                                                        amino                               G29   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            3-iodo-                                                                             H     N                                                                 phenyl                                                                        amino                                     G30   bond    N     C   CH.sub.3                                                                            CH.sub.3                                                                            4-iodo-                                                                             H     H                                                                 phenyl                                                                        amino                                     ______________________________________                                    

H. Group 8 Compounds

Group 8 compounds have the following chemical structure: ##STR8## whereR₂₆ and R₂₈ is independently alkyl, aryl, alkenyl, or alkynyl; and

R₂₇ is aryl. Examples of group 8 compounds are set forth in Table VIIIand shown in FIG. 1h.

                  TABLE VIII                                                      ______________________________________                                        Compound  R.sub.26   R.sub.27                                                                              R.sub.28                                         ______________________________________                                        H10       CH.sub.3   benzyl  3,4-                                                                          dihydroxyphenyl                                  H11       CH.sub.3   benzyl  2-hydroxyphenyl                                  H12       CH.sub.3   benzyl  3-hydroxyphenyl                                  H13       CH.sub.3   benzyl  4-hydroxyphenyl                                  H14       CH.sub.3   benzyl  3,4,5-                                                                        trihydroxyphenyl                                 ______________________________________                                    

I. Group 9 Compounds

Group 9 compounds have the following chemical structure: ##STR9## whereR₃₀ is either alkyl, alkenyl, or alkynyl, preferably CH₃ ; R₃₁ is aryl,preferably phenyl; and

R₃₂ is either O or S.

An example of a group 8 compound is I10, shown in FIG. 1i.

J. Group 10 Compounds

Group 9 compounds have the following chemical structure: ##STR10## whereR₃₃ is alkyl or aryl;

R₃₄, R₃₅, and R₃₆ are independently halogen, OH, hydrogen, alkoxy, SH,NH₂, or C(CH₃)₃, preferably R₃₄, R₃₅, and R₃₆ is independently selectedfrom H, OH, and C(CH₃)₃.

Examples of group 9 compounds are J10 and J11, shown in FIG. 1j.

k. Group 11 Compounds

Examples of Group 11 compounds are shown in FIG. 1k. Group 10 compoundsare identified by a "P."

l. Chemical Nomenclature

Definitions of some of the chemical groups mentioned in the applicationare described below.

An "alkyl" group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons, more preferably from 3 to 9 carbons.The alkyl group may be substituted or unsubstituted. When substitutedthe substituted groups is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂ or N(CH₃)₂, amino, SH, or aryl.

An "alkenyl" group refers to an unsaturated hydrocarbon group containingat least one carbon-carbon double bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkenyl group has 1to 12 carbons, more preferably from 3 to 9 carbons. The alkenyl groupmay be substituted or unsubstituted. When substituted the substitutedgroups is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, SH, or aryl.

An "alkynyl" group refers to an unsaturated hydrocarbon group containingat least one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons, more preferably from 3 to 9 carbons. The alkynyl groupmay be substituted or unsubstituted. When substituted the substitutedgroups is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, SH, or aryl.

An "alkoxy" group refers to an "--O-alkyl" group, where "alkyl" isdefined as described above.

An "aryl" group refers to an aromatic group which has at least one ringhaving conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups, all of which may be optionallysubstituted. The preferred substituents of aryl groups are hydroxyl,cyano, alkoxy, alkyl, alkenyl, alkynyl, amino, and aryl groups.

Carbocyclic aryl groups are groups wherein the ring atoms on thearomatic ring are carbon atoms. The carbon atoms are optionallysubstituted. Carbocyclic aryl groups include monocyclic carbocyclic arylgroups and optionally substituted naphthyl groups.

Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted.

A "carbalkoxy" group refers to a COOX group, wherein "X" is an loweralkyl group.

The term "lower" referred to herein in connection with organic radicalsor compounds respectively defines such with up to and including 7,preferably up to and including 4, and advantageously one or two carbonatoms. Such groups may be straight chain or branched.

II. Cell Proliferative Disorders

The described compositions and methods are designed to inhibit cellproliferative diseases by inhibiting PDGF-R activity. As discussedabove, proliferative disorders result in unwanted cell proliferation ofone or more subset of cells in a multicellular organism resulting inharm to the organism. Inappropriate PDGF activity can stimulate of cellproliferative disorders. Two ways in which inappropriate PDGF or PDGF-Ractivity can stimulate unwanted cell proliferation of a particular typeof cell are by directly stimulating growth of the particular cell, or byincreasing vascularization of a particular area, such as tumor tissue,thereby facilitating growth of the tissue.

The use of the present invention is facilitated by first identifyingwhether the cell proliferation disorder is PDGF-R driven. Once suchdisorders are identified, patients suffering from such a disorder can beidentified by analysis of their symptoms by procedures well known tomedical doctors. Such patients can then be treated as described herein.

Determination of whether the cell proliferation disorder is PDGF-Rdriven can be carried out by first determining the level of PDGF-Ractivity occurring in the cell or in a particular body location. Forexample, in the case of cancer cells the level of one or more PDGF-Ractivities is compared for non-PDGF-R driven cancers (e.g. A431 cells asdescribed below) and PDGF-R driven cancers (e.g., T98G glioblastomacells as described below). If the cancer cells have a higher level ofPDGF-R activity than non-PDGF-R driven cancers, preferably equal to orgreater than PDGF-R driven cancers, then they are candidates fortreatment using the described PDGF-R inhibitors.

In the case of cell proliferative disorders arising due to unwantedproliferation of non-cancer cells, the level of PDGF-R activity iscompared to that level occurring in the general population (e.g., theaverage level occurring in the general population of people or animalsexcluding those people or animals suffering from a cell proliferativedisorder). If the unwanted cell proliferation disorder is characterizedby a higher PDGF-R level then occurring in the general population thenthe disorder is a candidate for treatment using the described PDGF-Rinhibitors.

Cell proliferative disorders include cancers, blood vessel proliferationdisorders, and fibrotic disorders. These disorders are not necessarilyindependent. For example, fibrotic disorders may be related to, oroverlap with, blood vessel disorders. For example, atherosclerosis(which is characterized herein as a blood vessel disorder) results inthe abnormal formation of fibrous tissue.

A cancer cell refers to various types of malignant neoplasms, most ofwhich can invade surrounding tissues, and may metastasize to differentsites, as defined by Stedman's Medical Dictionary 25th edition (Hensyled. 1990). Examples of cancers which may be treated by the presentinvention include those intra-axial brain cancers, ovarian cancers,colon cancers, prostate cancers, lung cancers, Kaposi's sarcoma and skincancers, which have inappropriate PDGF-R activity. These types ofcancers can be further characterized. For example, intra-axial braincancers include glioblastoma multiforme, anaplastic astrocytoma,astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, meningioma,sarcoma, hemangioblastoma, and pineal parenchymal.

The formation and spreading of blood vessels, or vasculogenesis andangiogenesis respectively, play important roles in a variety ofphysiological processes such as embryonic development, wound healing andorgan regeneration. They also play a role in cancer development. Bloodvessel proliferation disorders refer to angiogenic and vasculogenicdisorders generally resulting in abnormal proliferation of bloodvessels. Examples of such disorders include restenosis, retinopathies,and atherosclerosis.

The advanced lesions of atherosclerosis result from an excessiveinflammatory-proliferative response to an insult to the endothelium andsmooth muscle of the artery wall. (Ross R., Nature 362:801-809 (1993).)Part of the response appears to be mediated by PDGF-BB secretion, andactivation of PDGF-R in endothelial and smooth muscle cells. Both cellmigration and cell proliferation play a role in the formation ofatherosclerotic lesions.

Fibrotic disorders refer to the abnormal formation of extracellularmatrix. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders.

Hepatic cirrhosis is characterized by the increase in extracellularmatrix constituents resulting in the formation of a hepatic scar.Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. Anincreased extracellular matrix resulting in a hepatic scar can also becaused by viral infection such as hepatitis. Lipocytes appear to play amajor role in hepatic cirrhosis. Inappropriate PDGF-R activity canstimulate lipocyte proliferation.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases, such asglomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes, transplant rejection, andglomerulopathies. PDGF has been implicated in the maintenance ofmesangial cell proliferation. (Floege, J. et al., Kidney International43S:47-54 (1993).)

As noted above, other such proliferative diseases can be identified bystandard techniques, and by determination of the efficacy of action ofthe compounds described herein.

A. Ovarian Cancer

One aspect of the invention relates to the treatment of ovarian cancer.Epithelial ovarian cancer accounts for nearly 90% of all ovarian tumorsand continues to be a highly lethal malignancy. Approximately 19,000 newcases of ovarian cancer are diagnosed in the United States annually, and12,000 of these women will die from the cancer (Rodriguez et al., inDeVita, Hellman, Rosenberg (eds) Biologic Therapy of Cancer, J BLippincott, 1991).

Treatment for advanced ovarian cancer generally includes cytoreductivesurgery followed by combination chemotherapy with alkylating agents suchas cisplatin and cyclophosphamide. However, long term survival ofadvanced ovarian cancer patients is extremely poor, in the range of10%-20%, principally because of the high incidence of metastatic tumorsthroughout the peritoneal cavity, and, in some cases, the lymph-nodes.Moreover, chemotherapy with cisplatin carries a potential for renaltoxicity and progressive neuropathy.

The invention reveals a pathological relationship between PDGF receptorexpression and epithelial ovarian cancer, and provides compositions andmethods for inhibiting inappropriate PDGF-R activity in epithelialovarian cancer cells to inhibit proliferation of the disease. Methods oftreating ovarian cancers comprise administering a composition whichinhibits inappropriate PDGF-R activity in ovarian carcinoma cells, insupporting stromal cells (i.e., the framework upon which a tumor ormetastatic lesion grows, including but not limited to connective tissueand vascular endothelial cells), and/or in associated vascularendothelial cells.

Ovarian cancers susceptible to treatment with the compounds describedherein include epithelial ovarian carcinoma, ovarian tumor metastases,and other cells of the ovary which express PDGF receptors. As describedbelow, compositions which inhibit PDGF-R activity also inhibitproliferation of ovarian cancer cells in vitro and inhibit the growth ofovarian tumors in vivo. More specifically, the use of one composition ofthe invention, A10, results in nearly complete inhibition of ovariantumor growth in mice xenotransplanted with human ovarian cancer cells,without significant cytotoxicity or mortality, thus providing a dramatictherapeutic effect.

Accordingly, as an example of the method of the invention, A10 isadministered to a patient diagnosed with ovarian cancer via any route ofadministration and in any suitable pharmaceutical carrier which resultsin bringing A10 in contact with PDGF receptor-positive ovarian cancercells and/or cells of the surrounding stroma. In view of the localizedspread of ovarian cancer throughout the peritoneal cavity, a preferredmethod of administration, particularly in advanced cases, is byintravenous or intraperitoneal injection of a non-toxic pharmaceuticalformulation of A10.

The preparation and use of therapeutically effective compositions fortreating ovarian cancers are described in detail in the sections whichfollow and by way of examples, infra. In addition to the compositionsspecifically disclosed herein, the invention provides for theidentification of other compositions which, because of their inhibitoryeffect on PDGF-R activity may be useful for inhibiting the proliferationof ovarian neoplasms. Candidate compositions may be identified by theirability to inhibit PDGF receptor autophosphorylation using any suitableassay, such as in vitro autophosphorylation inhibition ELISA andtyrosine kinase inhibition assays. Candidate compositions may beevaluated for therapeutic efficacy by testing their capacity to inhibitovarian cancer cell growth and, ideally, by testing inhibition ofxenografted tumors in vivo. The procedures described in the examples,infra, or similar procedures, may be employed for conducting such tests.

B. Glioma

Another aspect of the invention relates to the treatment of primaryintra-axial brain tumors of the glioma family, including, but notlimited to, astrocytomas and glioblastomae. Glioblastoma multiforme isthe most common and most malignant tumor of astrocytic origin in humanadults and accounts for more than half of all primary brain tumors (See,for example, Cecil Textbook of Medicine, Wyngaarden, Smith, Bennett(eds) W B Saunders, 1992, p. 2220).

Gliomas have the common property of direct invasive involvement of braintissue, are fundamentally malignant, and are inevitably fatal.Glioblastoma patients have a median survival time of less than one yeareven when treated aggressively with a combination of surgery,chemotherapy, and radiotherapy. Unfortunately, successful surgicalintervention is extremely rare in view of the difficulty orimpossibility of defining the microscopic borders of a glioma withinnormal brain tissue. Similarly, chemotherapy with alkylating agents hasmet with very little success, and no more than 10% of glioma patientsrespond significantly. Radiation therapy has demonstrated some value incontrolling the growth of gliomas, but often results in substantialneurologic impairment. Therapy with interferon-β, in combination withradiotherapy and chemotherapy, has met with some success (DeVita,Hellman, Rosenberg (eds) Biologic Therapy of Cancer, J B Lippincott,1991).

The invention reveals a pathological relationship between PDGF receptorexpression and glioma, and provides compositions and methods forinhibiting PDGF activity in glioma cells to inhibit proliferation of thedisease. Methods of treating gliomas comprise administering acomposition which inhibits PDGF-R activity expressed in glioma cellsand/or in proximate vascular endothelial cells. In particular, most ofthe compositions specifically disclosed herein are highly active atinhibiting PDGF receptor autophosphorylation in human glioma cells invitro. Several of these compositions inhibit the growth of culturedglioma cells, and one of these, A10, also inhibits the growth of variousglioma explant cultures. Moreover, A10 strongly suppresses the growth ofxenografted gliomas in mice; in some animals, tumor growth was inhibitedby greater than 95% relative to untreated controls.

Accordingly, as an example of the method of the invention, A10 isadministered to a glioma patient via any route of administration and inany suitable pharmaceutical carrier which will result in bringing A10 incontact with PDGF receptor-positive glioma cells, as well as proximatevascular endothelial cells, which typically proliferate in high gradegliomas. Intravenous and intra-arterial routes may be preferred routesof administration. In addition, recently-developed micro-cathetertechnology may be particularly effective at delivering the compositionsof the invention directly to the site of the glioma, thereby achievingimmediate localized contact with the cancer and proximate endothelialcells and possibly minimizing potential toxicity associated with moredistal intra-arterial delivery.

The preparation and use of therapeutically effective compositions forthe treatment of gliomas are described in detail in the sections whichfollow and by way of examples, infra. In addition to the compositionsspecifically disclosed herein, the invention provides for theidentification of other compositions which, because of their inhibitoryeffect on PDGF receptor activity, may be useful for inhibiting theproliferation of various intra-axial tumors. Candidate compositions maybe identified by their ability to inhibit PDGF receptor activity usingany suitable assay, such as in vitro autophosphorylation inhibitionELISA and tyrosine kinase inhibition assays. Candidate compositions maybe evaluated for therapeutic efficacy by testing inhibition of gliomacell growth and, ideally, by testing inhibition of xenografted tumors invivo.

III. A10

The present invention describes various compositions which can be usedto inhibit PDGF-R activity and thereby inhibit cell proliferationdisorders. The use of A10 to inhibit tumor growth in animalsdemonstrates the ability of these compositions to function in vivodespite various pharmacological considerations which are expected toprevent the composition from exerting its effect. Such in vivoinhibition is illustrated in the examples described below.

A10 is also known as leflunomide, HWA 486, and 5-methylisoxazole-4carboxylic acid-(4-trifluromethyl)-anilide. Various publications havediscussed different possibly uses of A10. According to the abstracts ofKommerer F-J, et al., U.S. Pat. No. 4,284,786 (1981) and Kommerer F-J,et al., U.S. Pat. No. 4,351,841 (1982), A10 "has antirheumatic,antiphlogistic, antipyretic and analgesic action, and can be used forthe treatment of multiple sclerosis." According to Talmadge J. E., andTwardzik D. R. Agents and Actions 35S:135-141 (1991), "the hypothesiswas suggested that the mechanisms of Leflunomide activity may be theinhibition of a cytokine specific kinase." Robertson S. M. and Lang L.S., European Patent Application 0 413 329 A2 (published 1991) which isconcerned with 5-methylisoxazole-4-carboxylic acids that encompassleflunomide, asserts:

The present invention is directed to methods for treating oculardiseases with immune etiology through the use of5-methyl-isoxazole-4-carboxylic acid anilides and hydroxyethlidene-cyanoacetic acid anilide derivatives. In addition the compounds are usefulfor treating ocular manifestation associated with systemic diseases withimmune etiology. The compounds exhibit immunosuppressive,antiinflammatory, and mild antiallergic activity and are useful for thetreatment of eye diseases such as uveitis (including rheumatoidnodules), retinitis, allergy (vernal keratocon junctivitis and allergicor giant papillar conjunctivitis) and dry eye (Sjogren's syndrome).Additionally the compounds are useful for prolonging graft survival ofcorneal or other ocular tissue and are useful as surgical adjuncts inpatients which are atopic or immune impaired.

The abstract of Barlett R. R. et al., entitled "Isoxazole-4-Carboxamidesand Hydroxyalklidene-Cyanoacetamides, Drugs Containing These Compoundsand Use of Such Drugs" PCT/EP90/01800, asserts:

Isoxazole-4-carboxamide derivatives and hydroxyalkylidene-cyanoacetamidederivatives are suitable for the treatment of cancer diseases. Thesecompounds can be prepared by prior art methods. Some of them are new andare suitable, in addition, for the treatment of rheumatic diseases.

Bartlett R. R. et al., Agents and Actions 32:10-21 (1991), asserts that"[l]eflunomide has been shown to be very effective in preventing andcuring several autoimmune animal diseases." Barlett also asserts that:

. . , we could show that tyrosine phosphorylation of the RR-SRC peptidesubstrate and the autophosphorylation of the epidermal growth factor(EGF) receptor were, dose dependently, inhibited by leflunomide.

Matter et al., FEBS 334:161-164 (November 1993)(not admitted to be priorart) describes the use of the active metabolite of leflunomide toinhibit EGF-dependent cell growth, including A431 cells. Matter alsoasserts:

Platelet-derived growth factor-dependent tyrosine phosphorylation wasalso inhibited by A77 1726 in intact cells at concentrations similar toEGF-dependent phosphorylation described in FIG. 3 (data not shown).

Studies on one composition of the invention, A10, described more fullyand by way of example infra, establish its potency against brain, lung,prostate, ovarian, skin, and colon cancer cells characterized byinappropriate PDGF-R activity rather than EGF activity. As illustratedin the examples described below A10 inhibits PDGF-R activity whilehaving little if any effect on EGF-receptor or HER2 phosphorylation. Inaddition, while A10 inhibited growth of tumors characterized byinappropriate PDGF-R activity, A10 did not significantly inhibit thegrowth of xenotransplanted cells expressing EGF receptor (A431epidermoid cells). This data is surprising in view of the resultsdescribed by Bartlett et al., supra, Agents and Actions in whichleflunomide was shown to inhibit EGF induced EGF receptorautophosphorylation and cell proliferation, and Mattear et al., supra,in which the active metabolite of leflunomide inhibited growth of A431cells.

The present disclosure demonstrates the ability of A10 to inhibitinappropriate PDGF-R activity and unwanted cell proliferation in vivo,such as occurs in cancers characterized by inappropriate PDGF-Ractivity. As illustrated by the examples described below, A10 can beused to selectively inhibit inappropriate PDGF-R activity

A compound is judged to effect phosphorylation if its ability to inhibitphosphorylation of a receptor (e.g., the IC₅₀ as described below) isless than its cytotoxic effect (e.g., the LD₅₀ as described below).Inhibition of phosphorylation of different receptor such as PDGF-R,EGF-R or HER-2 receptor is dependent on conditions such as drugconcentration. By "selectively inhibit" it is meant that a compound canbe used at a particular concentration to inhibit phosphorylation of thePDGF-R and have little if any effect on the phosphorylation of the EGF-Rand/or HER-2 receptor at the same concentration.

Preferably, the compound, like A10 can inhibit PDGF-R while havinglittle if any effect on EGF-R and/or HER-2 phosphorylation. By "littleif any effect" on EGF-R, or HER-2, activity is meant the receptoractivity is effected no more than 35%, more preferably, no more than20%, most preferably no more than 10%, at a particular receptor.

Tyrosine kinases are important in many biological processes includingcell growth, differentiation, aggregation, chemotaxis, cytokine release,and muscle contraction. Many of these events are mediated throughdifferent tyrosine kinase receptors. In addition, different tyrosinekinase receptors may be important for a particular biological functionin different cell types. By developing selective inhibitors of PDGF-Rthe possible toxic effect of the compound is decreased.

The compounds described herein vary in their ability to selectivelyinhibit PDGF-R. For example D14, G12, G13 and G14 inhibit PDGF-Rphosphorylation but do not effect EGF or Her-2 phosphorylation, whileC10 effects EGF-R, PDGF-R, and Her-2 phosphorylation.

IV. Mutated PDGF-R

Cell proliferative disorders characterized by inappropriate PDGF-Ractivity can also be inhibited using a mutated PDGFP-R. Ueno H., et al.,Science 252:844-252 (1991), describe nucleic acid encoding truncatedPDGF-R to inhibit PDGF-R phosphorylation in vitro. According to Ueno:

When truncated receptors were expressed in excess compared to wild-typereceptors, stimulation by PDGF of receptor autophosphorylation,association of phosphatidylinositol-3 kinase with the receptor, andcalcium mobilization were blocked.

Ueno did not demonstrate that inhibition of PDGF-R activity by a mutatedprotein could inhibit unwanted cell proliferation.

Such in vivo inhibition of unwanted cell proliferation is illustrated inthe examples described below using nucleic acid encoding the PDGF-Rhaving a stop codon just upstream from the first tyrosine kinase domain.The nucleic acid is used to introduce the truncated protein into a cell.For example, the nucleic acid encoding a truncated PDGF-R is placed intoa retroviral vector using standard recombinant DNA techniques. Thevector then infects a cell where its nucleic acid is ultimatelytranslated into protein producing a truncated PDGF-R. Other means ofintroducing the mutated protein into a cell include preparing themutated protein in vitro and introducing the protein into the cell witha vector, such as a liposome.

Mutant PDGF-R should be constructed to interfere with intermolecularphosphorylation that occurs between dimerized receptor. This can beaccomplished by various means such as 1) truncation of the PDGF-R,preferably to eliminate one tyrosine kinase domain, and most preferablyto eliminate both tyrosine kinase domains; 2) mutations which inhibitthe catalytic ability of the PDGF-R catalytic domain, such as mutationof lysine 602 to arginine which prevents the binding of ATP. Of thesemethods, mutation of tyrosine residues is preferred and truncation ofthe receptor is most preferred.

The use of nucleic acid encoding truncated PDGF-R to inhibit tumorgrowth in animals demonstrates the ability of such truncated receptorsto function in vivo despite various pharmacological considerations whichwould be expected to prevent the composition from exerting its effect.Thus, the present disclosure demonstrates that the use of nucleic acidencoding truncated PDGF receptor is not limited to inhibition of PDGF-Rin cell culture. Rather nucleic acid encoding PDGF-R can be used toinhibit inappropriate PDGF-R activity in animal cells thereby inhibitingthe growth of tumors in animal cells, and having application in otherPDGF-R related disorders.

V. Adminstration of Featured Compounds

The compounds of this invention can be administered to a patient alone,or in a pharmaceutical composition comprising the active compound and acarrier or excipient. The compounds can be prepared as pharmaceuticallyacceptable salts (i.e., non-toxic salts which do not prevent thecompound from exerting its effect).

Pharmaceutically acceptable salts can be acid addition salts such asthose containing hydrochloride, sulfate, phosphate, sulfamate, acetate,citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.(See e.g., supra. PCT/US92/03736). Such salts can derived using acidssuch as hydrochloric acid, sulfuric acid, phosphoric, acid and sulfamicacid, acetic acid, citric acid, lactic acid, tartaric acid, malonicacid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinine acid.

Pharmaceutically acceptable salts can be prepared by standardtechniques. For example, the free base form of the compound is firstdissolved in a suitable solvent such as an aqueous or aqueous-alcoholsolution, containing the appropriate acid. The salt is then isolated byevaporating the solution. In a another example, the salt is prepared byreacting the free base and acid in an organic solvent.

Carriers or excipient can be used to facilitate administration of thecompound, for example, to increase the solubility of the compound.Examples of carriers and excipient include calcium carbonate, calciumphosphate, various sugars or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols and physiologicallycompatible solvents. The compositions or pharmaceutical composition canbe administered by different routes including intravenously,intraperitoneal, subcutaneous, and intramuscular, orally, topically, ortransmuccosally.

Several of the featured compounds, such as A10 and B11, are hydrophobicand thus not very soluble in water. Effective doses of A10 can beobtained by using A10 in combination with PBTE:D5W. PBTE consists of asolution of 3% w/v benzyl alcohol, 8% w/v polysorbate 80, and 65% w/vpolyethylene glycol (MW=300 daltons) in absolute ethanol. PBTE:D5Wconsists of PBTE diluted 1:1 in a solution of 5% dextrose in water. Thesolubility of A10 in PBTE is about 60 mg/ml, and the solubility of A10in PBTE:D5W is about 5 mg/ml. The solubility of the other compoundsdescribed herein can be obtained using standard techniques. In addition,the active drug itself (e.g., B11) may be administered in an oralformulation.

Another way of overcoming the hydrophobicity problem includes the use offrequent small daily doses rather than a few large daily doses. Forexample, the composition can be administered at short time intervals,preferably the composition can be administered using a pump to controlthe time interval or achieve continuously administration. Suitable pumpsare commercially available (e.g., the ALZET® pump sold by Alzacorporation, and the BARD ambulatory PCA pump sold by Bard MedSystems).

Alternatively, prodrugs having increased solubility can be used.Prodrugs can break down into the active drug under physiologicalconditions. For example, Patterson et al., J. Med. Chem. 35:507-510(1992), describes A12(3-carboxy-5-methyl-N-[4(triflouromethyl)phenyl]4-isoxazolecarboxamide)which, like A10, can act as a prodrug for B11.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used, and the size andphysiological condition of the patient. For the treatment of cancers theexpected daily dose of A10 is between 1 to 2000 mg/day, preferably 1 to250 mg/day, and most preferably 10 to 150 mg/day. Drugs can be deliveredless frequently provided plasma levels of the active moiety aresufficient to maintain therapeutic effectiveness.

A factor which can influence the drug dose is body weight. Drugs shouldbe administered at doses ranging from 0.02 to 25 mg/kg/day, preferably0.02 to 15 mg/kg/day, most preferably 0.2 to 15 mg/kg/day.Alternatively, drugs can be administered at 0.5 to 1200 mg/m^(2/) day,preferably 0.5 to 150 mg/m² /day, most preferably 5 to 100 mg/m² /day.The average plasma level should be 50 to 5000 mg/ml, preferably 50 to1000 mg/ml, and most preferably 100 to 500 mg/ml. Plasma levels may bereduced if pharmacological effective concentrations of the drug areachieved at the site of interest.

VI. Administration of Mutated PDGF-R

The PDGF-R mutants can be administered as nucleic acid expressing theprotein, using standard techniques some of which are discussed below.Delivery vehicles include liposomes and other pharmaceuticalcompositions. Nucleic acid encoding a mutated PDGF-R can also beintroduced into a cell using standard techniques such as a retroviraland ion paired molecules. In those cases where the technique is carriedout ex vivo the cell is then put into a patient. Administration ofprotein is facilitated using a carrier or excipient as described above.

The specific delivery route of any selected agent depends on the use ofthe agent (such considerations are also applicable for theadministration of the featured compounds). Generally, a specificdelivery program for each agent focuses on agent uptake with regard tointracellular localization, followed by demonstration of efficacy.Alternatively, delivery to these same cells in an organ or tissue of ananimal can be pursued. Uptake studies include uptake assays to evaluate,e.g., cellular nucleic acid or protein uptake, regardless of thedelivery vehicle or strategy. Such assays also determine theintracellular localization of the agent following uptake, ultimatelyestablishing the requirements for maintenance of steady-stateconcentrations within the cellular compartment containing the targetsequence (nucleus and/or cytoplasm). Efficacy and cytotoxicity can thenbe tested. Toxicity not only includes cell viability but also cellfunction. Generally, the dosages of the mutated protein and nucleic acidis as described above for the featured compounds.

Drug delivery vehicles are effective for both systemic and topicaladministration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles increase thecirculation half-life of drugs which would otherwise be rapidly clearedfrom the blood stream. Some examples of such specialized drug deliveryvehicles falling into this category are liposomes, hydrogels,cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.Pumps can also be used for this purpose.

From this category of delivery systems, liposomes are preferred.Liposomes increase intracellular stability, increase uptake efficiencyand improve biological activity. Liposomes are hollow spherical vesiclescomposed of lipids arranged in a similar fashion as those lipids makingup the cell membrane. They have an internal aqueous space for entrappingwater soluble compounds and range in size from 0.05 to several micronsin diameter. Antibodies can be attached to liposomes to targetparticular cells.

Topical administration of PDGF-R mutants; and the featured compounds isadvantageous since it allows localized concentration at the site ofadministration with minimal systemic adsorption. This simplifies thedelivery strategy of the agent to the disease site and reduces theextent of toxicological characterization. Furthermore, the amount ofmaterial applied is far less than that required for other administrationroutes. Effective delivery requires nucleic acid to enter the cellmembrane or the cytoplasm of cells characterized by inappropriate PDGF-Ractivity and express the protein.

Agents may also be systemically administered. Systemic absorption refersto the accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes whichlead to systemic absorption include: intravenous, subcutaneous,intraperitoneal, intranasal, intrathecal and ophthalmic. Each of theseadministration routes expose the drug to an accessible diseased tissue.Subcutaneous administration drains into a localized lymph node whichproceeds through the lymphatic network into the circulation. The rate ofentry into the circulation has been shown to be a function of molecularweight or size.

VII. EXAMPLES

Examples are provided below to illustrate different aspects andembodiments of the present invention. These examples are not intended inany way to limit the disclosed invention. Rather, they illustratemethodology by which drugs having the disclosed formula can be readilyidentified by routine procedure to ensure that they have the desiredactivity. That is, compounds within the formula claimed herein can bescreened to determine those with the most appropriate activity prior toadministration to an animal or human. Other compounds can also bescreened to determine suitability for use in methods of this invention.

A description of some of the procedures used in the following examplesis described in the appendices below. The use of A10 in the differentprocedures is mentioned, however, compounds other than A10 were testedby these procedures by replacing A10 with the tested compound.

APPENDIX 1

1. Cell Lines

Cell lines were purchased from the ATCC unless otherwise specified.U1240 and U1242 cells were obtained from Dr. Joseph Schlessinger (NewYork University) and SF763 and SF767 cells were obtained from Dr.Michael Berens (Barrow Neurological Institute).

SF767T, SF763T, U118T, and SKOV3T are sublines of SF767, SF763, U118 andSKOV3 cells, respectively. They were derived by implanting the parentalcells SC into BALB/c, nu/nu mice. Tumors which displayed desirablegrowth characteristics were resected and finely minced in a sterilepetri dish. Two to five mL of appropriate medium was added to the slurryand the tumor pieces were further mechanically teased apart. Theresulting suspension was placed into tissue culture flasks and fed withthe appropriate culture medium supplemented with 100 unit/mL penicillinG sodium and 100 μg/mL streptomycin sulfate (Gibco, Grand Island, N.Y.).Medium was changed every two to three days. After three to fivepassages, the antibiotic supplements were removed and the cellsmaintained in antibiotic-free medium.

NIH3T3 mouse fibroblasts overexpressing the EGF receptor, Flk-1, IGF-1receptor or PDGF-b receptor were engineered using retroviral vectors.MCF7/HER2 cells were derived by overexpressing the HER2 gene usingretroviral constructs in an MCF7 background.

2. Cell Culture

All cell culture media, glutamine, and fetal bovine serum were purchasedfrom Gibco Life Technologies (Grand Island, N.Y.) unless otherwisespecified. All cells were grown in a humid atmosphere of 90-95% air and5-10% CO₂ at 37° C. All cell lines were routinely subcultured twice aweek and were negative for mycoplasma as determined by the Mycotectmethod (Gibco).

C6 cells were maintained in Ham's F10 supplemented with 5% fetal bovineserum (FBS) and 2 mM glutamine (GLN). T98G cells were cultured in MEMwith 10% FBS, 2 mM GLN, 1 mM sodium pyruvate (NaPyr) and non-essentialamino acids (NEAA). SKOV3T cells were cultured in DMEM, 10% FBS and 2 mMGLN.

NIH3T3 mouse fibroblasts engineered to overexpress Flk-1 or the EGFreceptor were maintained in DMEM containing 10% calf serum (CS) and 2 mMGLN. NIH3T3 cells engineered to overexpress the IGF-1 or insulinreceptor were maintained in DMEM containing 10% FBS and 2 mM GLN. HL60cells were maintained in RPMI 1640 with 10% FBS and 2 mM GLN. T47D andBT474 cells were maintained in RPMI 1640 with 10% FBS, GMS-G and 2 mMGLN.

DU 145 cells were grown in DMEM F12 with 10% FBS and 2 mM GLN. A172,A431, U118MG and RAG cells were grown in DMEM with 10% FBS and 2 mM GLN.L1210 cells were grown in DMEM with 10% horse serum and 2 mM GLN. C1300cells were grown in DMEM with 10% heat inactivated FBS, 2 mM GLN and 50mM β-mercaptoethanol. T98G, U138MG, U87MG, U373MG, U1240, U1242, Calu-3,Calu-6, SF767, SF767T, SF763, SF763T, SK-N-MC and SK-N-SH cells weregrown in MEM with 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN. MDA MB 361 andMDA MB 468 cells were grown in L15 with 10% FBS and 2 mM GLN. PC-3 cellswere grown in HAM'S F12 with 7% FBS and 2 mM GLN. A549 cells were grownin HAM'S F12 with 10%6 FBS and 2 mM GLN. ZR75-30 cells were grown inRPMI 1640 with 10% FBS, 2 mM GLN and 1 mM NaPyr. MCF7, MCF7/HER2, A375,BT549, 9L, C81-61, ZR 75-1 and K562 cells were grown in RPMI 1640 with10% FBS and 2 mM GLN. Ovcar3 cells were grown in RPMI 1640 with 20% FBS,2 mM GLN and 10 mg/mL insulin. D1B and T27A cells were grown in RPMI1640 with 10% heat-inactivated FBS, 2 mM GLN and 50 mMβ-mercaptoethanol; 7TD1 cells were grown in the same medium supplementedwith 50 units/mL of recombinant murine IL-6. Colo 320DM, WEHI-164.13,and HBL100 cells were grown in RPMI 1640 with 10% heat-inactivated FBSand 2 mM GLN. SKBR3 cells were grown in McCoy's 5A with 15% FBS and 2 mMGLN. PA-1 cells were grown in MEM with 10% heat-inactivated FBS, 2 mMGLN and NEAA. Neuro 2A cells were grown in MEM with 10% heat inactivatedFBS, 2 mM GLN, NEAA, NaPyr and 50 mM β-mercaptoethanol.

APPENDIX 2

1. Receptor Phosphorylation

The inhibition of receptor tyrosine kinase activity by A10 was studiedby western blot and ELISA procedures. For western blotting, cells wereplated in 2 mL growth medium into 6-well dishes (500,000 cells per well)and allowed to attach overnight. The medium was replaced with 2 mL MCDB105 (UCSF Cell Culture Facility) supplemented with 1% FBS. The plateswere then incubated overnight at 37° C., ambient CO₂. To test theeffects of compounds on ligand-mediated receptor autophosphorylation,cells were exposed to A10 or DMSO for 1 hr at 37° C. before thestimulation of receptor tyrosine kinase activity with ligand. After a 7min incubation at room temperature with ligand, the plates were put onice and washed three times with 1 mL ice-cold PBS plus 1 mMorthovanadate. Lysis was achieved by pipetting cells in 0.5 mL of buffercontaining 50 mM Tris, pH 7.4, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 mMsodium vanadate (Na₃ VO₄), 10 mM pyrophosphate, 1 mM PMSF, 10 mg/mLaprotinin, 10 mg/mL leupeptin. A 300 μL aliquot of each lysate wasimmediately added to 100 μL 4× Laemmli sample buffer (0.2 mM Tris pH6.9, 20% glycerol, 7% SDS, 5 mM EDTA, 5% β-mercaptoethanol) containingphosphatase inhibitors, 2 mM Na₃ VO₄ and 10 mM pyrophosphate. Sampleswere boiled for 5 min, frozen in dry ice-ethanol, and stored at -80° C.Proteins were resolved by SDS-PAGE (Bio-Rad Miniprotean II) andtransferred to nitrocellulose membrane (Schleicher & Schuell) at 120volts for 1 hr at room temperature in buffer containing 25 mM Tris pH8.3, 20% methanol, 0.2M glycine, 0.1% SDS. The integrity of theprotein-to-membrane transfer was determined by staining with 1% PonceauS in 5% acetic acid for 5 min. After destaining in several distilledwater rinses, the membrane was soaked overnight in blocking buffercontaining 5% milk in Tris-buffered saline, 0.05% Tween 20.Phosphotyrosine was detected by incubating the membrane (1 hr at roomtemperature) with an anti-phosphotyrosine antiserum diluted in blockingbuffer at 1:3000. For C6 cells, PDGF-b receptor content in the lysateswas confirmed in duplicate sample lanes by western blotting using anantibody specific for PDGF-b receptor (UBI). The antibodies werevisualized using ECL reagent from Amersham and by exposure to Fuji RXfilm.

For ELISA assays, cells were grown to 80-90% confluency in growth mediumand seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to30,000 cells per well. After overnight: incubation in 0.5%serum-containing medium, cells were changed to serum-free medium andtreated with test compound for 2 hr in a 5% CO₂, 37° C. incubator. Cellswere then stimulated with ligand for 5-10 min followed by lysis withHNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na₃ VO₄,0.2% Triton X-100, and 2 mM NaPyr). Cell lysates (0.5 mg/well in PBS)were transferred to ELISA plates previously coated withreceptor-specific antibody and which had been blocked with 5% milk inTBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100, ) atroom temperature for 30 min. Lysates were incubated with shaking for 1hr at room temperature. The plates were washed with TBST four times andthen incubated with polyclonal anti-phosphotyrosine antibody at roomtemperature for 30 min. Excess anti-phosphotyrosine antibody was removedby rinsing the plate with TBST four times. Goat anti-rabbit IgG antibodywas added to the ELISA plate for 30 min at room temperature followed byrinsing with TBST four more times. ABTS (100 mM citric acid, 250 mM Na₂HPO₄ and 0.5 mg/mL 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonicacid) plus H₂ O₂ (1.2 mL 30% H₂ O₂ to 10 mL ABTS) was added to the ELISAplates to start color development. Absorbance at 410 nm with a referencewavelength of 630 nm was recorded about 15 to 30 min after ABTSaddition. Cell lines used in ELISA assays included U1242 (PDGF-breceptor), HL-60 cells (GMCSF receptor/JAK-2), or NIH3T3 cellsoverexpressing the EGF receptor, Flk-1, IGF-1 receptor or the insulinreceptor. IC₅₀ values were estimated by comparing drug inhibition oftyrosine phosphorylation in the absence or presence of appropriateligand.

2. DNA Synthesis

The effects of A10 on PDGF-dependent DNA synthesis was determined bymeasuring the ³ H-thymidine incorporation into DNA of cells. Theconditions for the assay were essentially those described by Pollack, etal. J. Neurosurg., 73:106-112, 1990, with some modifications. T98G cellsin log phase growth were transferred to 96-well dishes at 20,000 cellsin 200 μL of serum-containing growth medium. After an overnightattachment period, the monolayers were washed twice with 200 μL MCDB 105medium and the cells were cultured in 200 μL serum-free MCDB 105 mediumfor 24 hr. Medium in wells was replaced with fresh medium alone (MCDB105 plus 5 μg/mL insulin), medium containing EPDGF-BB alone, or mediumcontaining PDGF-BB in combination with various concentrations of A10.The plates were incubated at 37° C. at ambient CO₂ for approximately 18hr. ³ H-thymidine (Amersham, 5 Ci/mmol) was added to each well to yielda final concentration of 5 μCi/mL and the plates were returned to 37° C.incubator. After 4 hr the medium was removed, the plates were put ontoice, and washed twice with 200 μL ice-cold PBS per well. Radioactivityincorporated into DNA was separated from unincorporated ³ H-thymidine byprecipitation with 100 μL ice-cold TCA for 10 min on ice. After twowashes with ice-cold TCA, the precipitate was solubilized (1% SDS in 100mL 10 mM Tris base) and transferred to liquid scintillation countingvials. Six mL of cocktail (Ready Safe, Beckman) was added andradioactivity quantified in a Beckman liquid scintillation counter modelLS6000SC.

3. Cell Cycle Analysis

NIH3T3 mouse fibroblasts overexpressing the human PDGF-b receptor wereseeded in DMEM supplemented with 10% CS and 2 mM GLN. Cells were grownto about 80% confluence and then treated overnight in serum-free medium(DMEM, 2 mM GLN, 2 mM NEAA, 2 mM NaPyr, and 2 mM HEPES). The cells wereincubated for 20 hr in the presence of PDGF-BB at 100 ng/mL and withvarious concentrations of A10 (0.1, 1, 10, 25, or 100 mM). Cells werethen collected, stained and analyzed by flow cytometry for DNA content.

4. Growth Assays

A10 was tested for inhibition of anchorage-dependent tumor cell growthusing the colorimetric assay described by Skehan, et al., J. Natl.Cancer Inst., 82:1107-1112, 1990. The assay measures protein content ofacid-fixed cells using the counterion binding dye sulforhodamine B (SRB,Sigma). A10 was solubilized in DMSO (Sigma, cell culture grade) anddiluted into appropriate growth medium at two-fold the desired finalassay concentration. In assays using C6 cells, A10 (100 μL) was added to96-well plates containing attached cellular monolayers (2000 cells/wellin 100 μL). For other cell lines, the cells (2000 cells/well in 100 μL)were introduced into wells immediately after dispensing the drugsolutions. After 4 days (37° C., 5% CO₂) the monolayers were washed 3times with PBS and fixed with 200 μL ice-cold 10% TCA (FisherScientific), and kept at 4° C. for 60 min. The TCA was removed and thefixed monolayers were washed 5 times with tap water and allowed to drycompletely at room temperature on absorbent paper. The cellular proteinwas stained for 10 min with 100 μL 0.4% SRB dissolved in 1% acetic acid.After 5 washes with tap water, the dye was solubilized in 10 mM Trisbase (100 μL per well) and absorbance read at 570 nm on a Dynatech platereader model MR5000. Growth inhibition data are expressed as apercentage of absorbance detected in control wells which were treatedwith 0.4% DMSO alone. DMSO controls were not different from cells grownin regular growth medium. IC₅₀ values were determined using a fourparameter curve fit function.

For the anchorage-independent tumor cell growth assay, cells (3000 to5000 per dish) suspended in 0.4% agarose in assay medium (DMEMcontaining 10% FCS) with and without A10 were plated into 35 mm dishescoated with a solidified agarose base layer (0.8% agarose). After a 2 to3 week incubation at 37° C., colonies larger than 50 μm were quantifiedusing an Omnicon 3800 Tumor Colony counter.

APPENDIX 3

1. Growth Assays for Tumor Cell Lines

For most tumor cell lines, inhibition of cell growth by A10 was assessedusing an SRB assay as described in Appendix 2.. For K562, DlB, L1210,7TDl, T27A, and Colo320 DM cells, an MTT assay was used to assess cellgrowth. (Hansen et al., J. Immunol. Methods, 119:203-210, 1989.)Briefly, 50 mL of growth medium containing various concentrations of A10and 50 mL of cell suspension (2,000 cells) were added to each well of a96-well plate. The cells were incubated at 37° C. for 4 days in ahumidified 7% CO₂ atmosphere. At the end point, 15 mL of MTT (5 mg/mL inPBS, Sigma) was added to each well. The plates were incubated at 37° C.for 4 hr followed by addition of 100 mL of solubilization solution (20%w/v of SDS in 50% N, N-dimethylformamide, pH 4.7) to each well. Theplates were incubated overnight in a sealed container with a humidifiedatmosphere. The absorbance was determined at 570 nm wavelength with areference wavelength of 630 nm using an ELISA plate reader.

2. Growth Assay for Primary Tumors

The effect of A10 on primary tumor growth was examined by Oncotech, Inc.(Irvine, Calif.). Viable tumors were placed into medium by thepathologist at the referring institution immediately after surgery, andshipped to Oncotech by overnight delivery. As soon as a tumor wasreceived, a portion was fixed in formalin for sectioning and theremainder trimmed of necrotic, connective and adipose tissues. All tumormanipulations were performed aseptically. The remaining tumor was placedinto a Petri dish containing 5 mL of medium (RPMI 1640 supplemented with10% FBS, 100 IU/mL penicillin, 100 mg/mL streptomycin and L-glutamine)and disaggregated mechanically with scissors into pieces 2 mm orsmaller. The resultant slurries were mixed with medium containing 0.003%DNase (2650 Kunitz units/mL) and 0.14% type I collagenase (enzymes fromSigma Chemical Co., St. Louis Mo.), placed into 50 mL flasks withstirring, and incubated for 90 min at 37° C. in a humidified 5% CO₂atmosphere. A portion of the cell suspension was used for cytospin slidepreparation and was examined after hematoxylin and eosin staining of thetissue sections by a medical pathologist to confirm the diagnosis, andto determine the tumor cell count and viability.

After enzymatic dispersion into a near single-cell suspension, tumorcells were filtered through nylon mesh, washed in medium, suspended insoft agarose (0.12%) and plated at approximately 20,000 cells per wellonto an agarose underlayer (0.4%) in 24-well plates. Cells wereincubated under standard culture conditions for five days in thepresence or absence of A10. Cells were pulsed with ³ H-thymidine(Amersham, 5 mCi per well) for the last 48 hr of the culture period.After the appropriate labeling period, tissue culture plates were heatedat 60° C. to melt the agarose, the cells harvested with amicro-harvester onto glass fiber filters and the radioactivitydetermined. Percent inhibition (PCI) was determined using the formula:PCI=1--(CPM treatment group Π CPM control group). Determinations ofcontrol group proliferation were performed in quadruplicate, whiletreatment group proliferation was determined in triplicate.

APPENDIX 4

1. Animals

Female athymic mice (BALB/c, nu/nu), BALB/c mice, Wistar rats and Fisher344 rats were obtained from Simonsen Laboratories (Gilroy, Calif.).Female A/J mice were obtained from Jackson Laboratory (Bar Harbor, ME).DA rats were obtained from B&K Universal, Inc. (Fremont, Calif.).Athymic R/Nu rats, DBA/2N mice, and BALB/c mice were obtained fromHarlan Sprague Dawley (Indianapolis, Ind.). Female C57BL/6 mice wereobtained from Taconic (Germantown, N.Y.). All animals were maintainedunder clean-room conditions in Micro-isolator cages with Alpha-dribedding. They received sterile rodent chow and water ad libitum.

2. Subcutameous Xenograft Model

Cell lines were grown in appropriate medium (see Appendix 1). Cells wereharvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at450×g for 10 min. Pellets were resuspended in sterile PBS or media(without FBS) to a particular concentration and the cells were implantedinto the hindflank of mice. Tumor growth was measured over 3 to 6 weeksusing venier calipers. Tumor volumes were calculated as a product oflength x width x height unless otherwise indicated. P values werecalculated using the Students' t-test. A10 in 50-100 μL excipient (DMSO,PBTE, PBTE6C:D5W, or PBTE:D5W) was delivered by IP injection atdifferent concentrations.

3. Intracerebral Xenograft Model

For the mouse IC model, rat C6 glioma cells were harvested and suspendedin sterile PBS at a concentration of 2.5×10⁷ cells/mL and placed on ice.Cells were implanted into BALB/c, nu/nu mice in the following manner:the frontoparietal scalps of mice were shaved with animal clippers ifnecessary before swabbing with 70% ethanol. Animals were anesthetizedwith isofluorane and the needle was inserted through the skull into theleft hemisphere of the brain. Cells were dispensed from HamiltonGas-tight Syringes using 30 gauge 1/2 inch needles fitted with sleevesthat allowed only a 3 mm penetration. A repeater dispenser was used foraccurate delivery of 4 μL of cell suspension. Animals were monitoreddaily for well-being and were sacrificed when they had a weight loss ofabout 40% and/or showed neurological symptoms.

For the rat IC model, rats (Wistar, Sprague Dawley, Fisher 344, orathymic R/Nu; approximately 200 g) were anesthetized by an IP injectionof 100 mg/kg Ketaset (ketamine hydrochloride; Aveco, Fort Dodge, Iowa)and 5 mg/kg Rompun (xylazine, 2% solution; Bayer, Germany). After onsetof anesthesia, the scalp was shaved and the animal was oriented in astereotaxic apparatus (Stoelting, Wood Dale, Ill.). The skin at theincision site was cleaned 3 times with alternating swabs of 70% ethanoland 10% Povidone-Iodine. A median 1.0-1.5 cm incision was made in thescalp using a sterile surgical blade. The skin was detached slightly andpulled to the sides to expose the sutures on the skull surface. A dentaldrill (Stoelting, Wood Dale, Ill.) was used to make a small (1-2 mmdiameter) burrhole in the skull approximately 1 mm anterior and 2 mmlateral to the bregma. The cell suspension was drawn into a 50 μLHamilton syringe fitted with a 23 or 25 ga standard bevel needle. Thesyringe was oriented in the burrhole at the level of the arachnoidea andlowered until the tip of the needle was 3 mm deep into the brainstructure, where the cell suspension was slowly injected. After cellswere injected, the needle was left in the burrhole for 1-2 minutes toallow for complete delivery of the cells. The skull was cleaned and theskin was closed with 2 to 3 sutures. Animals were observed for recoveryfrom surgery and anesthesia. Throughout the experiment, animals wereobserved at least twice each day for development of symptoms associatedwith progression of intracerebral tumor. Animals displaying advancedsymptoms (leaning, loss of balance, dehydration, loss of appetite, lossof coordination, cessation of grooming activities, and/or significantweight loss) were humanely sacrificed and the organs and tissues; ofinterest were resected.

4. Intraperitioneal Model

Cell lines were grown in the appropriate media as described inAppendix 1. Cells were harvested and washed in sterile PBS or mediumwithout FBS, resuspended to a suitable concentration, and injected intothe IP cavity of mice of the appropriate strain. Prior to implantationof 7TD1 cells, C57BL/6 mice were primed by IP injection of 0.5 mLPristane. SKOV3T cells were implanted into athymic mice without Pristanepriming. Mice were observed daily for the occurrence of ascitesformation. Individual animals were sacrificed when they presented with aweight gain of 40%, or when the IP tumor burden began to cause unduestress and pain to the animal.

5. Immunohistochemistry

Acetone-fixed, 5 μm frozen tissue sections from untreated xenografttumors derived from human, rat, or murine tumor cells were analyzed byimmunohistochemistry using highly specific receptor antibodies. Briefly,non-specific binding sites were blocked with 10% normal goat serum priorto the application of the primary antibody. Appropriate antibodyconcentrations were used to achieve the desired sensitivity andspecificity (rabbit anti-human PDGF-b receptor, 1:400 and rabbitanti-mouse Flk-1, 5.5 μg/mL). Tissue sections known to contain theprotein of interest served as positive controls. Appropriate negativecontrols of normal rabbit IgG and mouse anti-chicken IgG of the sameprotein concentration and isotype as the primary antibodies were used.The detection method was a three-step indirect procedure and consistedof the primary antibody bound to a biotin-labeled secondary antibody(goat anti-rabbit IgG 1:500) followed by streptavidin conjugatedhorseradish peroxidase.

The chromagen/substrate used was 0.05% diaminobenzidine/0.03% H₂ O₂.Tissue sections were counterstained with hemeatoxylin, dehydratedthrough ascending grades of ethanol, cleared in Xylene Substitute, andcoverslipped with Permount for microscopic evaluation. A +to +++ gradingsystem was used to identify the overall intensity of the expression with+=low, ++=moderate, and +++=high intensity. Specific staining reactionwas seen as either (T) tumor cell or (V) vascular endothelial cell orboth.

APPENDIX 5

1. In Vitro Immunology Assays

At the indicated times, animals were sacrificed, and the spleens wereaseptically removed and placed into sterile medium. Spleens wereprocessed into single cell suspensions by grinding between sterilefrosted glass microscope slides. After a single wash to remove tissuedebris, the spleen cells were resuspended in a hypotonic ammoniumchloride buffer to lyse erythrocytes. Lymphocytes were washed andresuspended to the appropriate concentrations in complete medium,consisting of RPMI plus 10% heat-inactivated FBS, 2 mM glutamine, 50 μMβ-mercaptoethanol, and penicillin-streptomycin. The responses of thelymphocytes were examined in the following assays according to acceptedprocedures (Current Protocols in Immunology. Coligan, J. E., Kruisbeek,A. M., Margulies, D. H., Shevach, E. M., Strober, W. (eds.) John Wileyand Sons, Inc., 1992).

1.a. Mitogen Responses

The T-cell mitogen, ConA, and the T-cell independent B-cell mitogen,LPS, were added to 96-well round-bottom wells at the indicatedconcentrations. Lymphocytes from normal, vehicle-dosed, and drug-dosedanimals were added at a final concentration of 2.5×10⁵ /well. Cultureswere usually set up in triplicate or quadruplicate. The plates wereincubated at 37° C. in a humidified atmosphere containing 5% CO₂ for theindicated times. Supernatants (approximately 100 μL) were carefullyremoved from the wells and stored at -80° C. for lymphokine andimmunoglobulin analyses. To measure proliferation of the lymphocytes, 1μCi of ³ H-thymidine was added to each well, and the plates wereincubated for 6 hr. The cultures were harvested onto glass fiberfilters, and the incorporated radioactivity was quantitated by liquidscintillation counting (Betaplate, Wallac).

1.b. Mixed Lymohocyte Responses

Lymphocytes from normal, vehicle-dosed, and drug-dosed animals wereplated at 2.5×10⁵ /well in round-bottom 96-well plates. Stimulator cellswere then added at the same cell concentration. The stimulator cellsconsisted of syngeneic or allogeneic lymphocytes which had been treatedwith 50 μg/mL of mitomycin C for 30 min prior to the assay. The plateswere incubated for 3 to 4 days, at which time supernatants wereharvested and the cultures were pulsed as described for the mitogenassays.

1.c. Lymphokine Assays

Supernatants from mitogen and MLR cultures were assayed for IL-2 andIL-6 content by the ability to support the growth of factor-dependentcell lines. HT-2 cells (10⁴ /well, IL-2-dependent) and 7TDl cells (2×10³/well, IL-6-dependent) were plated in 96-well flat-bottom plates, in 50μL/well. Supernatants were added in 50 μL/well, and the plates wereincubated overnight (HT-2) or four days (7TD1). Cellular proliferationwas determined in the MTT colorimetric assay (Appendix 3.1).

1.d. Immunoglobulin ELISA

Flat bottom 96-well EIA plates were coated with goat anti-mouse Igantibodies (Southern Biotechnology) overnight at 4° C. The plates wereblocked by the addition of PBS+1% BSA. After washing with PBS,supernatants from murine mitogen and MLR cultures were added andincubated at room temperature for 1 hr. The plates were washed with PBS,then HRP-labeled goat anti-mouse Ig antibodies were added and incubatedat room temperature for 1 hr. The plates were washed and developed bythe addition of substrate (ABTS).

APPENDIX 6

1. HPLC Assay

At specific times after treatment of mice or rats with A10, blood wascollected in heparinized tubes by terminal cardiac puncture. Plasma wasprepared and frozen in liquid nitrogen. Tissues and organs were resectedand immediately frozen in liquid nitrogen. After the addition of aninternal standard, plasma samples were acidified with HCl and extractedwith acetonitrile. The acetonitrile fraction was evaporated to drynessin a vacuum centrifuge with heating and redissolved in methanol. Tissueand organ samples were homogenized according to the weight ratio of 1:5(w:v) in 50 mM Tris-HCl, pH 7.4, at 20,000 rev/min, using a tissuehomogenizer (Brinkman Polytron Model PT3000). After the addition of aninternal standard, the homogenate was acidified with HCl and thenextracted with acetonitrile. The acetonitrile fraction was thenextracted with an equal volume of diethyl ether. The ether fraction wasevaporated to dryness in a vacuum centrifuge with heating andredissolved in methanol.

Samples for HPLC analysis were injected onto a Hewlett Packard Hypersilμ-pm C18 cartridge column (100×4.6 mm). The mobile phase was methanol:35mM KH₂ PO₄ (pH 4.5) 55:45 containing 4 mM triethylamine. The flow ratewas 1.2 mL/min. The compounds were monitored by UV absorption at 254 nmusing a Hewlett-Packard diode-array detector (HP Model 1090). Plasma andtissue concentrations were determine from standard curves using peakarea for quantitation. Plasma and tissue standard curves were preparedfrom plasma and tissue homogenates obtained from drug-free rats, andspiked with known amounts of A10 and B11. The results were corrected forrecovery of the internal standard. The internal standard used was5-methyl-pyrazole-4-carboxylic acid-(4-trifluoromethyl)-anilide.

APPENDIX 7

1. Effects of A10 on Body Weight

Athymic mice (BALB/c, nu/nu, female, 4-5 weeks old) received IPadministration of A10 (20 mg/kg/day) every day in 100 μL PBTE:D5W (1:1,v:v) for 101 days. Vehicle control animals received IP administration of100 μL PBTE:D5W (1:1, v:v) every day for 101 days, and untreated controlanimals received no treatments. There were eight animals in each group.Weights were measured on day 0 (one day prior to drug administration)and two times/week until experiment termination. The percent weightchange was calculated as mean weight at each determination as comparedto the mean weight on day 0.

2. Determination of LD₅₀

Groups of five to ten athymic mice (BALB/c, nu/nu, female), or BALB/cmice (male and female) were treated with A10 IP in 50 μL PBTE, 100 μLPBTE, or 50 μL DMSO. In one experiment, groups of five BALB/c female,mice received IP administration of Decadron® (dexamethasone sodiumphosphate for injection, 1.5 mg/kg) once per day for seven days prior toadministration of a single IP dose of A10. In an additional experiment,groups of five BALB/c female mice received IP injection of Dilantin®(phenytoin sodium for injection, 20 mg/kg) once per day for seven daysprior to administration of a single dose of A10. All animals wereobserved for 7 to 14 days after the last dose was administered. The LD₅₀was calculated from a plot of % mortality versus dose (log M) using afour parameter logistic equation with goat anti-mouse Ig antibodies(Southern Biotechnology) overnight at 4° C. The plates were blocked bythe addition of PBS+1% BSA. After washing with PBS, supernatants frommurine mitogen and MLR cultures were added and incubated at roomtemperature for 1 hr. The plates were washed with PBS, then HRP-labelledgoat anti-mouse Ig antibodies were added and incubated at roomtemperature for 1 hr. The plates were washed and developed by theaddition of substrate (ABTS).

Example 1 Inhibition of PDGF-R Autophosphorylation by A10

This example illustrates the ability of A10 to inhibit PDGF-Rautophosphorylation of rat C6 glioma cells. Rat C6 glioma cells (5×10⁵)were plated in MCDB105 medium containing 5% FC:3 in a 6-well plate andincubated for 24 hours at 37° C. The cells were then placed in mediawith 1% FCS for another 24 hours. The cells were treated with A10 at 50,100, or 200 mM for one hour at 37° C. The cells were then treated with20 ng/ml of PDGF-BB for 10 minutes at 37° C. The cells were lysed in 50mM Tris-HCl (pH 7.4) containing 2 mM EDTA, 10% glycerol, 1% NP-40, 1 mMNa+orthovanadate, 10 mM pyrophosphate, 1 mM PMSF, 10 mg/ml aprotinin and10 mg/ml leupeptin.

Proteins were then separated by SDS-polyacrylamide gel electrophoresis(PAGE). Proteins containing phosphorylated tyrosine were identified bywestern blotting with an anti-phosphotyrosine antibody. The level ofphosphorylated tyrosine was determined by quantitating the amount ofbound anti-phosphotyrosine. Quantitation was carried out by peak areaintegration using a Molecular Dynamics Computing Densitometer (Model300S), and Image Quant v3.0 software (Molecule Dynamics). Data wereexpressed as relative peak intensity (phosphorylation of a receptordivided by the total amount of phosphorylated tyrosine).

PDGF-BB stimulated autophosphorylation of the PDGF-R, while A10inhibited such stimulation. Increasing concentrations of A10 resulted inreduced PDGF stimulated receptor phosphorylation. A10 at a concentrationof 200 mM reduced PDGF-R phosphorylation below that occurring in theabsence of PDGF-BB stimulation.

Example 2 Selective Inhibition of PDGF-R Autothosphorylation by A10

A10 inhibits autophosphorylation of the PDGF-R in human T98Gglioblastoma cells, while having little if any effect onautophosphorylation of the EGF receptor. T98G cells were plated inMCDB105 medium containing 2% FBS and incubated for 24 hours at 37° C.The media was aspirated and then replaced with MCD3105 and the cellswere treated for one hour with 200, 500 or 1,000 mM A10. Cells weretreated with different concentrations of A10 (0, 200, 500 and 1000 mM)and in the presence or absence of ligand. The cells were then treatedwith ligand for 10 minutes (20 ng/ml PDGF-BB or 50 ng/ml EGF). The cellswere lysed and the level of phosphorylated receptor was quantitated asdescribed in Example 1. A10 inhibited autophosphorylation of PDGF-R byPDGF, but had little if any effect on the ability of EGF to stimulateautophosphorylation of EGF-R.

Example 3 Inhibition of PDGF-R Phosphorylation by Various Compounds

This example illustrates the ability of various compounds to inhibitPDGF-stimulated receptor phosphorylation. U1242 MG cells were plated in96-well plates at a concentration of 5×10⁴ cells/well in cultured mediacontaining 0.5% FBS. The cells were incubated for 24 hours. The cellswere then treated with a particular compound for 2 hours followed by theaddition of 100 ng/ml PDGF-BB and incubation for 10 minutes.

Cells were lysed in 0.2M Hepes, 0.15 M NaCl, 10% V/V glycerol, 0.04%Triton X-100, 5 mM EDTA, 5 mM Na+vanadate and 2 mM Na⁺ pyrophosphate.Cell lysates were then added to an ELISA plate coated with an anti-PDGFreceptor antibody (Genzyme). ELISA plates were coated at 0.5 mg ofantibody/well in 150 ml of PBS for 18 hours at 4° C. prior to theaddition of the lysate.

The lysate was incubated in the coated plates for 1 hour and then washedfour times in TBST (35 mM Tris-HCl pH 7.0, 0.15 M NaCl, 0.1% TritonX-100). Anti-phosphotyrosine antibody (100 ml in PBS) was added and themixture was incubated for 30 minutes at room temperature. The wells werethen washed four times in TBST, a secondary antibody conjugated to POD(TAGO) was added to each well, and the treated wells were incubated for30 minutes at room temperature. The wells were then washed four times inTBST, ABTS/H₂ O₂ solution was added to each well and the wells wereincubated for two minutes. Absorbance was then measured at 410 nm.

The cytotoxicity of each drug was also determined. The cells were platedas described above. Following incubation with drug, cell survival wasmeasured by an MTT assay as described by Mossman J. Immunol. Methods65:55-63 (1983), or by measuring the amount of LDH released(Korzeniewski and Callewaert, J. Immunol. Methods 64:313 (1983); Deckerand Lohmann-Matthes, J. Immunol. Methods 115:61 (1988).

The results are shown in Table IX. IC₅₀ valves (i.e., the dose requiredto achieve 50% inhibition) were determined using the ELISA screeningassay. LD₅₀ values (i.e., the dosage which results in 50% toxicity) weredetermined using an MTT or LDH assay.

IC₅₀ values for inhibiting PDGF-stimulated receptor phosphorylation inU1242 cells ranged from 0.4 to >500 mM. As seen in Table IX most of thecompounds tested inhibited PDGF-stimulated receptor phosphorylation. Inall cases inhibition of receptor phosphorylation was not due tonon-specific effects on cell viability as shown by the higher LD₅₀.Thus, these drugs are good candidates for compounds which can be used totreat cell proliferative diseases by inhibiting PDGF-R activity. G13 andG14 had the lowest IC₅₀ but had a LD₅₀ less than A10. Generally, thepreferred compounds are those having the highest therapeutic index (LD₅₀/IC₅₀), which is a measure of the safety index.

                  TABLE IX                                                        ______________________________________                                                ELISA     CYTOXICITY                                                            P-TYR       LDH       MTT                                                     U1242       U1242     U1242                                         Compound  IC50(mM)    LD50(mM)  LD50(mM)                                      ______________________________________                                        A10       65          >500      700                                           B10       180                                                                 B12       100         >500      >1351                                         B13       180                                                                 B14       180                                                                 B15       120                   200                                           B16       35                    50                                            B17       125         >1000     >500                                          B18       160                                                                 B19       100                                                                 C10       25                    >500                                          C11       70          >500      >500                                          D11       8           >441      90                                            D12       60          >386      >390                                          D13       30          >500      >500                                          D14       20          >100      >500                                          D15       20          400       80                                            D16       50          >168      >167                                          D17       >100                                                                E10       45                                                                  E11       90                                                                  E12       180                                                                 E13       >100                                                                E14       100                                                                 E15       5                     >100                                          E16       125                                                                 F10       45                                                                  F11       100                                                                 F12       70                                                                  G10       10          >485      >490                                          G11       15          90        145                                           G12       10          >333      >333                                          G13       0.4         >100      100                                           G14       0.8         >100      >500                                          G15       100                                                                 G16       35                    >100                                          G17       100                                                                 G18       10          >100                                                    G19       90                                                                  G20       >100                                                                G21       6           >100                                                    G22       1           >100                                                    H12       30                                                                  I10       90          >317      >320                                          ______________________________________                                    

Example 4 A10 Inhibits PDGF-stimulated DNA Synthesis and Cell CycleProgression

This example illustrates the ability of A10 to inhibit PDGF-BBstimulated DNA synthesis and cell cycle progression. The effect of A10on DNA synthesis in T98G cells in the absence or presence of PDGF-BB wasdetermined by measuring ³ H-thymidine incorporation into DNA. Thepercentage of cells in the S phase of the cell cycle was determined byflow cytometry.

Cells were cultured as described in the appendices above. The assayconditions were essentially those described by Pollack et al., J.Neurosurg. 73:106-112 (1990) with some modifications. Cells (rat C6 orhuman T98G) in log phase growth were transferred to 96-well dishes at2×10⁴ cells in 200 ml MCDB 105 medium containing 2% FBS. After anovernight attachment period the media was changed to serum free assaymedia (MCDB 105 with 5 mg/ml insulin) and the cells were incubated for18-24 hours.

DNA synthesis studies were initiated by adding 50 ng/ml of PDGF-BB aloneor in combination with various concentrations of A10. The effect onbasal ³ H-thymidine incorporation was determined in the absence of PDGF.The plates were incubated at 37° C. for approximately 18 hours. ³H-thymidine (Amersham, 5 Ci/mmol) was added to each well to yield afinal concentration of 5 mCi/ml, the plates were returned to the 37° C.incubator, after 4 hours the medium was removed and the plates were puton ice. Each well was then washed twice with 200 ml ice-cold PBS.Radioactivity incorporated into DNA was separated from unincorporated ³H-thymidine by precipitation with 100 ml ice-cold TCA for 10 minutes.After two washes with ice-cold TCA, the precipitate was solubilized (1%SDS in 100 ml 20 mM Tris-base) and transferred to liquid scintillationcounting vials. Six ml of cocktail (Ready Safe, Beckman) was added andradioactivity quantified in a Beckman liquid scintillation counter modelLS6000 SC.

A10 decreased PDGF-stimulated DNA synthesis in both types of cells,however a greater effect was seen in human T98G glioblastoma cells thanrat C6 glioma cells. To confirm these results, the effect of A10 onPDGF-stimulated entry into the S phase of the cell cycle was examined.NIH3T3 cells engineered to overexpress the human PDGF-b receptor weregrowth-arrested (serum-starved) followed by treatment with PDGF ligandin the presence or absence of A10. The cells were analyzed for DNAcontent by flow cytometry. The results of this analysis are summarizedin FIG. 3. Treatment with PDGF resulted in a marked increase in cellsresiding in S phase (62%) relative to cells not treated with PDGF (11%).However, cells treated with A10 showed a dose-dependent decrease in thenumber of cells progressing to the S phase of the cell cycle in responseto PDGF, indicating that PDGF-stimulated mitosis was blocked by A10.

These results contrast with the results of a similar experiment in whichA10 at a concentration of 100 mM was not able to inhibit EGF-stimulatedmitosis in NIH3T3 cells overexpressing the human EGF receptor (FIG. 3).These results confirm the selectivity of A10 for PDGF-mediatedsignaling.

Example 5 Inhibiting the Activity of Different Receptor Kinases

The ability of different compound to inhibit different receptor tyrosinekinases was tested. The testing results are shown in Table X.

                  TABLE X                                                         ______________________________________                                                   PDGFR   EGFR      HER2  FLK-1                                                 IC50    IC50      IC50  IC50                                       Compound   (μM) (μM)   (μM)                                                                             (μM)                                    ______________________________________                                        G14        0.8 (W) NT        NT    >30 (W)                                    G25        0.9     >50       >50   >50                                        J10        1 (W)   >100      >100  NT                                         P13        1.1     >50       >50   >50                                        G13        1.5     NT        >50   >10 (W)                                    P10        3 (W)   31        >100  NT                                         J11        3       >100      >50   >25                                        F10        5 (W)   NT        NT    >50                                        G24        5       >100      >50   9.3                                        D11        8 (W)   NT        NT    >50                                        G10        10 (W)  NT        NT    >50                                        G12        10 (W)  NT        NT    >50                                        G18        10 (W)  550 (W)   NT    NT                                         E14        10 (W)  NT        NT    >50                                        G22        14      >100      >50   4.4                                        D20        14      >50       >50   >50                                        C13        16      16        29    3.3                                        D15        19 (W)  NT        NT    >50                                        G11        20 (W)  NT        NT    NT                                         G29        23      <0.8      34    10                                         C10        25 (W)  NT        NT    NT                                         G23        25 (W)  >100      >100  325.2                                      H10        25 (W)  NT        NT    >50                                        H12        25 (W)  NT        NT    NT                                         P12        25 (W)  NT        <50   >50                                        P14        26      >50       10    >200                                       D13        30 (W)  NT        NT    >50                                        P25        30 (W)  >100      >100  >500                                       G30        32      >100      34    10                                         D18        34      >50       >50   >50                                        P15        46      >100      >50   >50                                        D14        47      >100      >50   >50                                        G27        48      >100      >50   >50                                        ______________________________________                                    

Example 6 In Vitro Efficacy

The efficacy of A10 as a direct growth inhibitor was determined on tumorcell lines, and primary tumors isolated from patients. The effects ofA10 on tumor cell lines were determined by exposing cells to a range ofdrug concentrations and quantitating cell density after 4 days using theprocedures described in Appendix 3. Table XI provides the results oftesting different cell lines.

                  TABLE XI                                                        ______________________________________                                        Effects of A10 on Growth of Various Tumor Types                                                         IC.sub.50                                           Tumor Type     Cell line  (μM)                                             ______________________________________                                        glioma         SF763T     0.8                                                                SF767T     3                                                                  U1242      19                                                                 A172       32                                                                 T98G       62                                                                 U87MG      78                                                                 SF767      87                                                                 SF763      110                                                                U373MG     115                                                                U118MG     150                                                                U1240      250                                                                U138MG     >400                                                ovarian        SKOV3T     40                                                                 PA-1       40                                                                 SKOV3      >100                                                               Ovcar3     >100                                                breast         BT474      >100                                                               MCF7/HER2  116                                                                MDA MB 468 150                                                                T47D       195                                                                MDA MB 361 200                                                                MCF7       288                                                                ZR75-30    300                                                                ZR 75-1    355                                                                HBL100     >400                                                               BT549      >400                                                               SKBR3      >400                                                lung           Calu-6     70                                                                 A549       118                                                                Calu-3     >400                                                prostate       PC3        46                                                                 DU145      >100                                                melanoma       A375       25                                                                 C81-61     40                                                  colon          Colo 320DM 34                                                  epidermoid     A431       34                                                  leukemia       K562       26                                                  ______________________________________                                    

The IC₅₀ values for A10 ranged from 0.8 μM up to >400 μM.

The effects of A10 on in vitro growth of primary tumor cells isolatedfrom six patients with glioblastoma multiformae (GBM) and six patientswith ovarian carcinoma were tested. Specimens were obtained from newlydiagnosed and previously untreated patients and evaluated as describedin Example 13, Appendix 3.

A positive correlation between inhibition of tumor growth by A10 andPDGF-R expression was observed for both tumor types. As theconcentration of A10 increased (from 0 to 400 mM) tumor cell growthdecreased for both types of tumors. The growth inhibition wasdose-dependent for both tumor types and varied among the tumor cells.the IC₅₀ values ranged from 39 μM to 198 μM for the GBM tumors and from20 mM to 140 mM for the ovarian tumors.

Example 7 In Vivo Efficacy Studies Using A10 and B11

This example summarizes several experiments illustrating the ability ofA10 to inhibit the growth of different tumors in vivo. The first seriesof experiments looks at effects of different formulation and treatmentregimens. The second series of experiments looks at the effects of A10on a variety of different tumors.

Different Formulations and Treatment Regimens

C6 and SKOV-3(T) cells were grown in culture, as described in "cellgrowth" above, and implanted into the hind flank of a female Balb/cnu/nu mouse at 3×10⁶ cells (for C6 experiments), or 1×10⁷ cells (forSKOV-3 experiments) in 100 ml of PBS on Day 0. U87MG, U118MG or U373MGhuman glioblastoma cells (obtained from the ATCC), or A4312 were alsoimplanted into athymic mice. Mice implanted with tumors, andnon-implanted mice were administered A10 or B11 via intraperitonealinjection in a volume of 50 ml of DMSO, 100 ml PBTE:D5W, or 100 ml PBTEbeginning on Day 1 or as otherwise indicated. Tumors were measured usingvenier calipers and tumor volume was calculated as a product of tumorlength, width, and height.

In one set of experiments mice were implanted with A431, rat C6 gliomacells, SKOV-3(T) ovarian tumor cells and treated with 15 mg/kg/day ofA10 (DMSO). Tumor growth progressed logarithmically in untreated, andDMSO controls. In contrast, tumor growth progressed only slightly (i.e.,greater than 90% inhibition in tumor growth after 20 days compared withcontrol) in A10 treated animals implanted with rat C6 glioma cells orSKOV-3(T) ovarian tumor cells. A10 had little (i.e. no more than 25%)effect on A431 tumor growth. Tumor growth of mice implanted with rat C6glioma cells was inhibited with 15 mg/kg/day of B11 (DMSO) to the sameextent as implanted mice treated with 15 mg/kg/day of A10 (DMSO).

In another set of experiments mice implanted with C6 glioma cells weretreated with A10. Table XII summarizes the ability of A10 to inhibit ratC6 glioma cells in athymic mice using different treatment regimens. Thepercent inhibition refers to size of the tumor from A10 treated animals,divided by the size of the tumor from vehicle control treated animals.The different treatment regimens resulted in inhibition of 51% togreater than 95%.

                  TABLE XII                                                       ______________________________________                                        A10 Dosing Regimen Studies                                                    Dose          Regimen  % Inhibition                                           ______________________________________                                        20 mg/kg      daily    >95%                                                   (PBTE:D5W)                                                                    20 mg/kg      2 days   77%                                                    (DMSO)                                                                        20 mg/kg      4 days   60%                                                    (DMSO)                                                                        30 mg/kg      2 days   91%                                                    (DMSO)                                                                        30 mg/kg      3 days   87%                                                    (DMSO)                                                                        40 mg/kg      2 days   >95%                                                   (PBTE)                                                                        60 mg/kg      weekly   51%                                                    (PBTE)                                                                        100 mg/kg     weekly   63%                                                    (PBTE)                                                                        ______________________________________                                    

In another set of experiments the affects different A10 dosing regimenswere examined on C6 glioma cells. A10 was administered IP at differentdoses using different regimens beginning one day post-implatation. Thetotal dose of A10 administered to the animals was compared to percentinhibition. These studied showed that higher doses of A10administratered less frequently have anti-tumor efficacy equivalent tothat seen with lower doses administered daily provided that the totaldose adminstratered is equal. For example, 95% inhibition of tumorgrowth could be achieved by the administration of A10 to mice at 20mg/kg every day, 40 mg/kg every two days, or 80 mg/kg every four days.C6 cells (3×10⁶ cells) were implanted SC into the hindflanks of BALB/c,nu/nu mice

In another set of experiments the affect of different doses of A10 onglioblastoma were determined. Table XIII presents data illustrating theability of A10 to inhibit glioblastoma cells in vivo.

                  TABLE XIII                                                      ______________________________________                                        Cell          Dose                                                            Line          (mg/kg) % Inhibition                                            ______________________________________                                        U87           5       52                                                                    10      58                                                                    15      66                                                                    20      92                                                      U118          15      57                                                      U373          15      54                                                      SF763T        20      89                                                      SF767T        20      70                                                      ______________________________________                                    

The percent inhibition refers to tumor size in treated animals versustumor size in untreated animals.

Table XIV compares the efficacy of different A10 formulations in vivo(mice implanted with C6 cells). PBTE, PBTE:D5W and DMSO formulationsshowed equivalent in vivo inhibition of tumor growth.

                  TABLE XIV                                                       ______________________________________                                        Efficacy vs. Formulation                                                      Dose           Formulation                                                                             % Inhibition                                         ______________________________________                                        15 mg/kg/day   DMSO      90%                                                  20 mg/kg/day   DMSO      95%                                                  15 mg/kg/day   PBTE      92%                                                  20 mg/kg/day   PBTE      >95%                                                 40 mg/kg/2     PBTE      >95%                                                 days                                                                          20 mg/kg       PBTE:D5W  >95%                                                 ______________________________________                                    

The effects of A10 on animal mortality, using DMSO, PBTE, or PBTE:D5Wformulations is presented in Table XV (mice implanted with C6 cells).PBTE:D5W formulations significantly reduced the mortality rate comparedto DMSO formulations, and PBTE formulations.

                  TABLE XV                                                        ______________________________________                                        Effects of A10 on Mortality                                                   Dose       Treatments    Mortality                                                                              n                                           ______________________________________                                        20 mg/kg/day                                                                             21            54%      26                                          (DMSO)                                                                        20 mg/kg/day                                                                             27-100        0%       80                                          (PBTE:D5W)                                                                    25 mg/kg/day                                                                             67            0%       8                                           (PBTE:D5W)                                                                    20 mg/kg/day                                                                             20-48         8%       12                                          (PBTE)                                                                        30 mg/kg/day                                                                             48            50%      4                                           (PBTE)                                                                        40 mg/kg/day                                                                             48            75%      4                                           (PBTE)                                                                        ______________________________________                                    

Inhibition of Different Tumor Types by A10

This section describes experiments comparing the ability of A10 toinhibit the growth of different types of tumors. In one set ofexperiments the ability of A10 to inhibit ovarian, melanoma, prostate,lung, and mammary tumor cell lines established as SC xenografts wasexamined using the procedures described in the appendices. In a secondset of experiments the effects of A10 on the growth of murine leukemiacell lines in a synthetic model were tested as using the proceduresdescribed in the appendices above.

Table XVI summarizes the results of the studies using SC xenografts. Atdoses ranging from 12 to 20 mg/kg/day, A10 effectively inhibited thegrowth of glioma, SKOV3T (human ovarian), PA-1 (human ovarian), A375(human melanoma), PC-3 (human prostate), Calu-6 (human lung), and D1Band L1210 (murine leukemias). However, A10 failed to significantlyinhibit the growth of A549 (human lung), MCF7 (human mammary) and A431(human epidermoid) xenografts.

                  TABLE XVI                                                       ______________________________________                                        Effect of A10 on Tumor Growth                                                                           Dose   % Inhibition                                                                          P <                                  Tumor type                                                                            Cell line                                                                              Strain   mg/kg/day                                                                            (day)** ***                                  ______________________________________                                        glioma  C6*      nu/nu    20     >95 (21)                                                                              0.00001                                      C6*      nu/nu.sup.†                                                                     20     84 (18) 0.00001                                      9L*      nu/nu    20     83 (20) 0.00001                                      U87MG    nu/nu    15     75 (28) 0.0092                                       U118T    nu/nu    15     57 (47) 0.0027                                       U373MG   nu/nu    15     54 (37) 0.0477                                       SF763T   nu/nu    20     85 (22) 0.00001                                      SF767T   nu/nu    20     70 (22) 0.00001                              ovarian SKOV3T   nu/nu    15     94 (21) 0.0014                                       PA-1     nu/nu    20     53 (36) 0.04                                 melanoma                                                                              A375     nu/nu    20     53 (31) 0.03                                         A375     SCID.sup.¥                                                                         15     35 (31) 0.002                                prostate                                                                              PC-3     nu/nu    20     71 (45) 0.01                                         PC-3     SCID.sup..English Pound.                                                                12                    0.001                        lung    Calu-6   nu/nu    20     64 (28) 0.0001                                       A549     SCID.sup.¶                                                                   15     19 (48) NS                                   leukemia                                                                              D1B*     DBA/2    20     95 (22) 0.00001                                      L1210*   DBA/2    20     75 (18) 0.04                                 epidermoid                                                                            A431     nu/nu    15     40 (15) NS                                   mammary MCF7     SCID.sup..INTEGRAL.                                                                    15     8 (27)  NS                                   ______________________________________                                    

Table XVI. Tumor cells were implanted SC into the indicated strains ofmice. Daily treatment with vehicle or A10 was initiated on day onepost-implant, with the following exceptions: †=day 4, ¥=day 9, .EnglishPound.=day 15, ¶=day 29. ∞=day 8. *D1B and L1210 are murine tumor celllines, C6 and 9L are rat tumor cell lines; all others are human tumorcell lines. **Data are presented as percent inhibition of tumor growthon the day indicated as compared to vehicle control; n=8 to 10mice/group except D1B and L1210 where n=4 mice/group. ***P values werecalculated by Student's t-test; NS=not significant.

In the second set of experiments, A10 was found to significantlyincrease the survival of animals bearing SKOV3T IP Xenografts and micebearing 7DT1 iosgrafts. In one experiment, SKOV3T cells (2×10⁶ cells)were implanted into the IP cavity of BALB/c, nu/nu mice. A10 wasadministered IP in 50 μL DMSO at 15 mg/kg/day for 21 days beginning oneday post-implantation and mice were monitored for survival (8 animalsper treatment and control group). All animals in the control group diedafter 27 days, while one of A10 treated animals died by 28 days and 40%of A10 treated animals were alive after 32 days.

In another experiment, 7TD1 (B-cell hybridoma) cells were implanted IPin syngeneic immunocompetent C57BL/6 mice and the animals were treatedfor 30 days with 15 mg/kg/day of A10 (8 animals per treatment andcontrol group). A10 was administered IP in 50 μL DMSO at 15 mg/kg/dayfor 30 days beginning one day post-implantation. Dosing ceased on day30, and surviving mice were observed for an additional 50 days. Allanimals in the control group died by day 9, while 3 of 8 animals in theA10-treated group survived past day 80.

Example 8 Targeting Cancer Characterized by Inappropriate PDGF-RActivity

This example illustrates the ability of A10 to inhibit cancerscharacterized by inappropriate PDGF-R activity while having little or noeffect on tumors not characterized by PDGF-R activity. PDGF-R expressionwas measured qualitatively using a western blot. SRB assays assessedcell growth by determining total cellular protein using sulforhodamine-B(Skehan, T et al., J. Natl. Cancer Inst. 82:1107-1112 (1990). Soft agarassays (SAA) were carried out by seeding cells at colony density in asemi-solid agar medium onto a base layer of agar after two to threeweeks the size and number of colonies were quantitated with an automatedOmincon 3800™ tumor colony counter. SRB and SAA values are expressed asIC₅₀ values in mM. In vivo inhibition was determined in xenograftedathymic mice. The results are shown in Table XVII.

                  TABLE XVII                                                      ______________________________________                                        RECEPTOR EXPRESSION vs. GROWTH INHIBITION                                               PDGF-R                                                              Cell Line Expression                                                                              SRB        SAA  In Vivo                                   ______________________________________                                        C6        ++        0.3        0.4  95%                                       SF767     -         100        ND   18%                                       SF767T    ++        3          0.5-2                                                                              78%                                       SF763     -         >100       ND   35%                                       SF763T    ++        ND         ND   89%                                       SKOV-3    -         >100       6.3  ND                                        SKOV-3T   ++        50         0.3  95%                                       ______________________________________                                    

As seen in Table XVII growth inhibition is strongest on cells expressinghigh levels of PDGF-R, establishing a clear link between receptoractivity and cancer cell proliferation.

Example 9 Post Implant In Vivo Inhibition Using A10

This example describes the effects of A10 when administered to mice anumber of days after cancer implantation. In one experiment, SCID micecontaining PC-3 prostate cell line were treated with A10 starting 15days after tumor transplant. The weekly treatment comprised 12 mg/kg/dayof A10 (PBTE:D5W) for five days and two days of no treatment. The micewere treated for three week. In another experiment, mice containing A375melanoma were treated in the same way as those implanted with PC-3except 15 mg/kg/day of A10 was used and treatment began 9 days aftertumor implant.

In both cases, tumor growth was inhibited by A10. PC-3 tumor growth wasinhibited about 50% after three weeks. A375 melanoma growth wasinhibited about 40% after three weeks. Thus, this example furtherillustrates the utility of A10 to inhibit tumor growth by showing itsability to inhibit tumor which have been growing in a host prior totreatment (Cf. Example 7 where treatment of tumor began one day aftertransplant).

Example 10 Effect of A10 on Intracerebral Tumor Growth

The effect of A10 on tumor growth in brain tumor models was examined ina series of experiments.

In two separate experiments A10 was administered to athymic micefollowing intracerebral (IC) implantation of C6 cells, the averagesurvival time of A10-treated animals was significantly increasedcompared to controls. The results of this experiment are shown in FIG.4).

In another experiment, in which A10 was administered to athymic micefollowing IC implantation of U87MG cells, the mean survival of theA10-treated animals was 65 days compared to a mean survival of 60 daysin control animals an increase that was not significant (P=0.15).

The efficacy of A10 was also tested in an IC model in athymic rats. Asshown in FIG. 5, A10 had a slight but significant positive effect on thesurvival of athymic rats following the IC implantation of C6 tumorcells.

Example 11 Immunology Studies

The effects of A10 on several parameters of normal immune function,including proliferation of lymphocytes, generation of cytotoxic effectorcells, lymphokine production, and immunoglobulin secretion, wereexamined. These studies involved in vivo treatment of rats and mice withA10, followed by in vitro analyses of immune function. A detailedsummary of the methods used in the immunology studies are described inAppendix 5.

Effects of in vivo Administration of A10 on Immune Function of NormalMice

Naive BALB/c mice were treated with 15 mg/kg/day of A10 or vehicle(PBTE:D5W) for 7 and 21 days. The animals were sacrificed and theirspleen cells were assayed in vitro for responses to ConA (a T-cellmitogen), LPS (a T-cell independent B-cell mitogen), and alloantigens(C3H/HeJ spleen cells) as described in Appendix 5. The IL-2, IL-6, Igcontent and cellular proliferation were measured after 48 hrs (mitogens)or 72 hrs (alloantigens). The results; are summarized in Table XVIII.

                  TABLE XVIII                                                     ______________________________________                                        Effects of A10 on Normal Immune Function in Naive Mice                                                           .sup.3 H-                                         IL-2    IL-6      Ig        thymidine                                         production                                                                            production                                                                              production                                                                              uptake                                     Stimulus d7     d21    d7   d21  d7   d21  d7   d21                           ______________________________________                                        ConA     0      0      ++   0    0    0    0    0                             LPS      0      0      0    0    +    +/-  0    0                             Allo     0      0      0    0    0    0    0    0                             ______________________________________                                    

Table XVIII. Female BALB/c mice were treated with A10 for 7 or 21 days.Spleen cells were removed and stimulated in vitro with mitogens oralloantigens. Cellular proliferation, cytokine production, and Igproduction were assayed as described in Appendix 5. Results arepresented as a comparison between drug-treated and vehicle-treated(PBTE:D5W) animals. 0=no change; ±=slight increase in some animals;+=moderate increase in all animals; ++=strong increase in all animals.

Two parameters appeared to be affected by A10 treatment: ConA-inducedIL-6 production was higher in the A10-treated group than in controls atday 7, but this difference was not apparent at day 21. LPS-induced Igproduction was slightly higher in the A10 group than in controlsfollowing 7 days of treatment, but this difference was not apparent in 3of 4 mice treated with drug for 21 days. These data indicate that theeffects of A10 on IL-6 production and Ig production by splenocytes weretransient even with continued administration of A10.

Effects of in vivo Administration of A10 on Immune Function of MiceDuring a Primary Immunization

The effect of A10 in animals responding to an active primaryimmunization was also examined. BALB/c mice were immunized SC with 50 μgkeyhole limpet hemocyanin (KLH) suspended in complete Freund's adjuvant(CFA); control mice were mock-immunized with an emulsion ofphosphate-buffered saline (PBS) in CFA. Beginning on day 1, mice weredivided into three treatment groups: untreated, vehicle (PBTE:D5W) only,or A10 at 20 mg/kg/day. On day 14, all mice were sacrificed, and theirspleen cells were assayed. In addition, immune responses to KLH andtetanus toxoid were measured as described in Appendix 5.

Cytokine production (IL-2 and IL-6) by splenocytes was not significantlyaffected by vehicle alone or drug as summarized in Table XIX. However,A10 treatment was associated with a slight inhibition of theproliferative responses of spleen cells after restimulation with theimmunizing antigen (KLH) in vitro, (Table XIX). Similar effects wereseen when the spleen cells were stimulated with LPS or allogeneic cells(Table XIX).

                  TABLE XIX                                                       ______________________________________                                        Effects of A10 on Normal Immune Function in Mice                              Responding to a Primary Immunization                                                    IL-2     IL-6       Ig     .sup.3 H                                 Stimulus  production                                                                             production production                                                                           uptake                                   ______________________________________                                        KLH       0        0          0      --                                       ConA      0        0          0      0                                        LPS       0        0          0      --                                       Allo      0        0          0      --                                       ______________________________________                                    

Table XIX. BALB/c mice were immunized with KLH, then divided into threetreatment groups; dosing began on day 1 and continued until day 14post-immunization. Spleen cells from untreated, vehicle-treated, orA10-treated mice, as well as non-immune control mice (normal) werecultured with the indicated mitogens or alloantigens. Cellularproliferation, cytokine production, and immunoglobulin production wereassayed as described in Appendix 5. Results are presented as acomparison of the responses of drug-treated animals to those ofvehicle-treated (PBTE:D5W) controls. 0=no change; -=slight decrease inall animals.

Effects of in vivo Administration of A10 on Immune Function ofTumor-Bearing Mice

BALB/c mice received SC implants of syngeneic WEHI-164.13 fibrosarcomacells on day 0. On day 1, the mice were divided into three treatmentgroups: untreated, daily treatment with vehicle alone (PBTE:D5W), ordaily treatment with A10 at 20 mg/kg/day. On day 17, the animals weresacrificed and their spleens were removed. The spleen cells were assayedin vitro for responses to alloantigens and mitogens. Treatment with A10did not affect the proliferation, cytokine secretion, or Ig secretion ofspleen cells stimulated with alloantigen or mitogens as summarized inTable XX.

The spleen cells were also assayed for the generation ofWEHI-164.13-specific cytotoxic T-lymphocytes (CTL) in the mixedlymphocyte tumor cell culture (MLTC). Spleen cells from untreatedtumor-bearing animals mounted a vigorous CTL response (44% specificlysis at E:T=50:1). This response was inhibited almost completely inanimals receiving the vehicle alone (13% specific lysis). The vehiclehas an ethanol content of ˜15% and ethanol is known to transientlyeffect CTL generation and activity. (Walia, et al., Proc. Soc. Exp.Biol. Med., 192:177-200, 1989.)

Treatment with A10 enhanced the CTL response (78% specific lysis)compared to vehicle-treated animals; the CTL response of spleen cellsfrom A10-treated mice was also higher than that from untreated controlanimals. Thus, it appears that in this immunocompetent mouse model, A10dosing overcomes the inhibitory effect of the vehicle in the generationof CTL.

                  TABLE XX                                                        ______________________________________                                        Effects of A10 on Normal Immune Function in Mice                              Bearing a Primary Tumor                                                                 IL-2     IL-6       Ig     CTL                                      Stimulus  production                                                                             production production                                                                           activity                                 ______________________________________                                        Tumor     0        0          0      ++                                       ConA      0        0          0      NT                                       LPS       0        0          0      NT                                       Allo      0        0          0      NT                                       ______________________________________                                    

Table XX. Tumor-bearing mice were treated with vehicle or A10, or leftuntreated, for 17 days post-implantation. Mice were sacrificed andspleen cells were cultured with the indicated mitogens, alloantigens, ormitomycin C-treated WEHI-164.13 tumor cells. Cellular proliferation,cytokine production, immunoglobulin production, and CTL activity wereassayed as described in Appendix 5. Results are presented as acomparison of the responses of drug-treated animals to those ofvehicle-treated (PBTE:D5W) controls. 0=no change; ++=strong increase inall animals; NT=not tested.

Effects of in vivo Administration of AlG on Immune Function of Rats

Wistar rats received SC implants of C6 cells on day 0. Beginning on day1, and daily thereafter, the rats received IP doses of vehicle(PBTE:D5W) or A10 at 8.4 mg/kg/day (equivalent to 20 mg/kg/day of A10 inmice). The control group was not treated after tumor implantation. Onday 21, the animals were sacrificed and the spleens were removed for invitro analyses. Splenocytes were stimulated with ConA, LPS, andalloantigen (DA rat spleen cells) as in the murine studies. Of theparameters measured, only cellular proliferation in the syngeneic mixedlymphocyte response (MLR) appeared to be affected by A10 treatment assummarized in Table XXI. The syngeneic MLR of the untreated,vehicle-treated, or tumor-bearing animals was much higher than that ofthe non-tumor bearing animals. This proliferative response was reducedin one of four A10-treated tumor-bearing animals to a level similar tonon-tumor bearing controls. The allogeneic response was not reduced.

The spleen cells from Wistar rats were also assayed for the generationof C6-specific CTL in the MLTC and the results summarized in Table XXI.Spleen cells from tumor-bearing, untreated animals mounted a vigorousCTL response (35% specific lysis at E:T=100:1), while cells fromnon-tumor bearing control rats mounted a weak: response (13% specificlysis at E:T=100:1). Similar to the studies in BALB/c mice, it appearedthat the vehicle inhibited the generation of CTL in a 7-day culture. Incontrast to the murine studies however, A10 did not overcome theinhibitory effect of the vehicle in immunocompetent rats.

                  TABLE XXI                                                       ______________________________________                                        Effects of A10 on Normal Immune Function in Rats                              Bearing a Primary Tumor                                                                                     .sup.3 H-                                                 IL-2     IL-6       thymidine                                                                            CTL                                      Stimulus  production                                                                             production uptake activity                                 ______________________________________                                        Tumor     0        0          0      0                                        ConA      0        0          0      NT                                       LPS       0        0          0      NT                                       Syn       0        0          +/-    NT                                       Allo      0        0          0      NT                                       ______________________________________                                    

Table XXI. Wistar rats received SC implants of C6 cells, followed bydaily treatment with vehicle or A10; tumor-bearing, untreated animalsand normal, non-tumor-bearing animals were included in the study. On day21, animals were sacrificed and their spleen cells were stimulated withthe indicated mitogens (ConA, LPS), syngeneic (Syn) or alloantigens(Allo), or mitomycin C-treated C6 tumor cells. Cellular proliferation,cytokine production, and CTL activity were assayed as described inAppendix 5. Results are presented as a comparison of the responses ofdrug-treated animals to those of vehicle-treated (PBTE:D5W) controls.0=no change; ±=decrease in some drug-treated animals to levels detectedin normal (non-tumor-bearing, non-drug-treated) animals; NT=not tested.

The results of these immunology studies indicate that A10 does notadversely affect the normal immune responses of imnunnocompetent orimmunodeficient rodents during or following in vivo dosing. Furthermore,the efficacy of the drug when tested against murine tumor cell lines insyngeneic immunocompetent hosts indicates that A10 does not impairanti-tumor immunity.

Example 12 Pharmacology of A10

Several pharmacological properties of A10 have been studied in mice andrats including its metabolism and half-life in plasma and brain tissue.A detailed description of the methods utilized in studying thepharmacology of A10 can be found in Appendix 6. In addition, thepharmacokinetic profile of A10 was studied in conjunction with formaltoxicity studies of the compound in rats and monkeys.

Metabolism of A10

The isoxazole ring of A10 undergoes an intramolecular rearrangement to2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide (B11). Themetabolism of A10 to B11 was investigated ex vivo in fresh heparinizedplasma from humans or rats. The conversion of A10 to B11 was monitoredusing reverse-phase HPLC as described in Appendix 6. At 25° C. (ambienttemperature), A10 underwent complete conversion to B11 by 21 hr in humanplasma. A similar experiment was performed using rat plasma withincubation at 37° C.; A10 was completely converted to B11 after a 2 hrincubation. The conversion of A10 to B11 does not occur inheat-inactivated plasma (15 min at 60° C.) from humans or rats.Additionally, both B11 and A10 together were incubated with fresh humanplasma and the concentrations monitored. No A10 was detected after 21hr; only B11 was present.

The metabolite, B11, appears to be equipotent to A10 in in vitro growthassays as well as equipotent in inhibiting PDGF-stimulated DNA synthesisand PDGF-stimulated cell cycle progression. B11 is also equipotent inthe inhibition of the growth of C6 tumors in vivo. These data suggestthat B11 is an active metabolite of A10. The use of A10 to treat cellproliferative disorders is preferred because it is more suitable thanB11 for formulation as an IV solution.

Pharmacokinetics of A10 and B11 in vivo

The pharmacokinetics of A10 and its metabolite (B11) were investigatedin vivo after intraperitoneal (IP) administration of A10 to mice andrats. When athymic (BALB/c, nu/nu, female) mice received A10 (120 mg/m²,40 mg/kg), the drug was converted to B11 at 3 hr post-dose, while theconcentration of the parent compound was below the limits of detectionin the systemic circulation. B11 was detected in plasma for more than 48hr and had a t_(1/2) of 16 hr and T_(max) of 3 hr. The area under theconcentration time curve (AUC) for B11 was calculated to be 2773μg.h/mL. The clearance rate (CL) was calculated to be 0.29 mL/hr,assuming 100% bioavailability and mean input time is zero. The volume ofdistribution (VD) was calculated to be 6.7 mL. In the brains of thesemice, B11 exhibited a t_(1/2) of 14 hr and a T_(max) of 3 hr.

                                      TABLE XXII                                  __________________________________________________________________________    Pharmacokinetics of A10 and B11 in Mice and Rats After IP                     Administration                                                                                                 Wistar                                                                  BALB/c                                                                              rats                                                  athymic mice      mice  80 mg/kg                                              40 mg/kg                                                                            75 mg/kg                                                                            75 mg/kg                                                                            40 mg/kg                                                                            560                                                   120 mg/m.sup.2                                                                      225 mg/m.sup.2                                                                      225 mg/m.sup.2                                                                      120 mg/m.sup.2                                                                      mg/m.sup.2                                            single                                                                              single                                                                              1/week ×                                                                      single                                                                              single                                                dose  dose  4     dose  dose                                         __________________________________________________________________________    Plasma                                                                        t.sub.1/2  (hr)                                                                     B11                                                                              16    15.5  16.9  14.9  6                                                  A10                                                                              <1.5  <1    <1    ND    <2                                           T.sub. max (hr)                                                                     B11                                                                              3     3     3     3     6                                                  A10                                                                              <0.5  ND    ND    ND    <2                                           C.sub. max                                                                          B11                                                                              111 ± 14                                                                         249 ± 26                                                                         190 ± 12                                                                         208 ± 34                                                                         260                                                A10                                                                              ND    ND    ND    ND    5.0                                          AUC      2773  5660  5012  4444  6417                                         (μg · hr/mL)                                                      CL (mL/hr)                                                                             0.29  0.27  0.30  0.18  0.26                                         VD (mL)  6.7   6.4   8.2   3.1   5.3                                          Brain                                                                         t.sub.1/2  (hr)                                                                     B11                                                                              14    NT    NT    2     6                                                  A10                                                                              <1.5  NT    NT    <1    20                                           T.sub.max (hr)                                                                      B11                                                                              3     NT    NT    1     6                                                  A10                                                                              1.0   NT    NT    1.0   7                                            __________________________________________________________________________

Table XXII. Female athymic mice (BALB/c, nu/nu; four per group) weretreated IP with A10 as indicated. BALB/c mice (female, four per group)were treated IP with 120 mg/m² (40 mg/kg) A10. Wistar rats (female, oneper time point) were treated IP with 560 mg/m² (80 mg/kg) A10. Plasmaand brain samples were prepared and analyzed by HPLC as described inAppendix 6. All results were calculated by the method of internalstandardization. NT=not tested, ND=not detected.

The pharmacokinetic profile was also determined in athymic mice for theA10 dose calculated to be the LD₁₀. Intraperitoneal administration ofA10 at 225 mg/m² (75 mg/kg) resulted in the detection of B11 in plasmafor more than 48 hr (the last time point analyzed) with a t_(1/2) of15.5 hr and T_(max) of 3 hr (Table XXII). The AUC was calculated to be5660 μg.hr/mL. The CL was calculated to be 0.27 mL/hr, assuming 100%bioavailability and mean input time is zero. The VD was calculated to be6.4 mL.

The pharmacokinetic profile of the A10 LD₁₀ (75 mg/kg) was also studiedin mice after four IP treatments given once every 7 days. B11 wasdetected in plasma for 48 hr after the last dose and had a t_(1/2) of16.9 hr and T_(max) of 3 hr (Table XXII). The AUC for B11 was calculatedto be 5012 μg.hr/mL. The CL was calculated to be 0.3 mL/hr, assuming100% bioavailability and mean input time is zero. The VD was calculatedto be 8.2 mL.

The pharmacokinetic profile of A10 was determined in BALB/c mice. BALB/cmice received a single IP dose of A10 (120 mg/m², 40 mg/kg). A10 hadbeen completely converted to B11 and was undetectable by 1 hr followingdrug administration. B11 was detected in plasma for more than 48 hr (thelast time point analyzed) and had a t_(1/2) of 14.9 hr and T_(max) of 3hr as summarized in Table XXII. The AUC was calculated to be 4444μg.hr/mL. The CL was calculated to be 0.18 mL/hr, assuming 100%bioavailability and mean input time is zero. The VD was calculated to be3.1 mL. In the brains of these mice, B11 exhibited a t_(1/2) of 2 hr anda T_(max) of 1 hr (Table XXII).

When rats (Wistar, female) received a single IP dose of A10 (560 mg/m²,80 mg/kg) the drug was converted to B11 by 2 hr post dose, the firsttime point analyzed. However, A10 was detectable up to 8 hr post-dose.As summarized in Table XXI, A10 had a t_(1/2) of <2 hr and T_(max) of <2hr. B11 was detected for greater than 24 hr post-dose and had a t_(1/2)of 6 hr and T_(max) of 6 hr. The AUC was calculated to be 6417 μg.hr/mL.The CL was calculated to be 0.26 mL/hr, assuming 100% bioavailabilityand mean input time is zero. The VD was calculated to be 5.3 mL. In thebrains of these rats, B11 exhibited a t_(1/2) of 6 hr and a T_(max) of 6hr while A10 exhibited a t_(1/2) of 20 hr and a T_(max) of 7 hr (TableXXII).

In vivo Plasma Levels

In vivo plasma levels of B11 were determined in athymic mice aftereither a single dose or repeated daily doses of A10 and the results aresummarized in Table XXII. Three hours (at the T_(max)) after a singleadministration of A10 (20 mg/kg), the B11 concentration was determinedto be 51.2±7.3 μg/mL (range 38.8-55.2 μg/mL) while A10 was not detected.The plasma steady-state levels of B11 were also determined in athymicmice after repeated administration (20 mg/kg/day) of A10 at 24 hrintervals (8 administrations). The steady-state maximum plasma level ofB11 was 79.7±11.2 μg/mL (range 65.2-94.6 Ag/mL), and the steady-stateminimum or trough level was 33.6±7.3 μg/mL (range 23.4-45.8 μg/mL). Theplasma level of B11 after achieving steady-state (maximum steady-statelevel) was 64% greater than after a single dose (Table XXII).

                  TABLE XXII                                                      ______________________________________                                        Plasma Levels of B11                                                                 B11 Concentration (μg/mL)                                                    Single      Multiple                                                 A10 Dose Dose        Doses     % Increase                                     ______________________________________                                        20 mg/kg 51.2 ± 7.3                                                                              79.7 ± 11.2                                                                         64                                             40 mg/kg 110.9 ± 15.3                                                                           161.1 ± 27.8                                                                         69                                             ______________________________________                                    

Table XXII. Plasma levels of B11 were determined after single ormultiple daily administrations of A10 at 20 or 40 mg/kg/day in athymicmice. All data are the mean of 4 animals.

The B11 plasma concentration at T_(max) was also determined after asingle administration of A10 at 40 mg/kg. The B11 concentration was110.9±15.3 μg/mL (range 93.5-131.3 μg/mL) while A10 was not detected.The plasma steady-state levels of B11 were also determined in athymicmice after repeated administration (40 mg/kg) of A10 at 24 hr intervals(6 administrations). The steady-state maximum plasma level of B11 was161.1±27.8 μg/mL (range 119.9-204.1 μg/mL), and the steady-state minimumor trough level was 38.2±21.7 μg/mL (range 13.5-66.9 μg/mL). The plasmalevel of B11 after achieving steady-state was 69% greater than after asingle dose (Table XXII). These data demonstrate that B11 accumulates inplasma after multiple daily administrations of A10.

In addition to plasma levels, concentrations of A10 and B11 weredetermined in brain tissue after administration of A10 to rats and mice.The results are summarized in Table XXIV. The brain levels of B11 andA10 differed between both species and strains. The highest level of B11in the brain was detected in athymic mice, followed by BALB/c mice, andWistar rats respectively. At 2 and 4 hr post-dose, there was 11 and 6.5times, respectively, more B11 in the brains of athymic mice compared toWistar rats. At 2 hr post-dose, there was 2 times more A10 in the brainsof athymic mice as compared to Wistar rats; at 4 hr post-doseapproximately equivalent amounts of A10 were detected in the brains ofathymic mice and Wistar rats.

                                      TABLE XXIV                                  __________________________________________________________________________    Comparison of B11 and A10 Levels in Brain Tissue                              B11 and A10 Concentration in Brain Tissue (ng/mg)                             Time Post-                                                                          Athymic mice                                                                              BALB/c mice Wistar rats                                     dose  B11   A10   B11   A10   B11  A10                                        __________________________________________________________________________    2 hr  21.9 ± 12.7                                                                      19.9 ± 13.5                                                                       3.5 ± 2.8*                                                                       9.1 ± 13.5*                                                                     1.9 ± 1.5                                                                       8.9 ± 4.0                               4 hr  21.3 ± 19.3                                                                      1.8 ± 1.6                                                                        10.7 ± 3.7                                                                       3.8 ± 2.9                                                                        3.3 ± 1.5                                                                       2.1 ± 3.2                               __________________________________________________________________________

Table XXIV. B11 and A10 levels in brain tissue were determined asdescribed in Appendix 6. *Determined at 2.5 hr post-dose. n=4 animals.

These pharmacology studies demonstrate that A10 is metabolized to B11 inboth mice and rats. In addition, A10 and B11 can be detected in bothplasma and brain tissue. However, there appears to be a difference inboth the pharmacokinetics and distribution of A10 and B11 to plasma andbrain between the two species examined.

Example 13 Preliminary Toxicology Studies Using A10

Preliminary toxicology studies of A10 included testing the effect of A10on blood cells, body weight, LD₅₀ determinations. To determine theeffect of potential ancillary medications on the LD₅₀ of A10,combination experiments were also performed. A detailed description ofthe methods used can be found in Appendix 7. The pharmacology andtoxicology studies illustrate that A10 can be administered to animalsunder conditions having little if any adverse effect on the animal,particularly when PBTE:D5W formulations are used. Other suitableformulations can be obtained by one skilled in the art using thisapplication as a guide.

Effect of A10 on Blood Cells

Many cancer therapeutics are cytotoxic in nature and have profoundeffects on blood cells resulting in cytopenia. The effects of A10 on thenumber of red blood cells, white blood cells, and the percent oflymphocytes versus polymorphonuclear cells were examined.

A10 at 15 mg/kg/day, did not appear effect blood differentials over a 21day period for drug delivered in DMSO, PBTE or PBTE:D5W (1:1). Drugdelivered in PBTE:D5W at 20 or 25 mg/kg/day did not affect the number ofRBCs, WBCs or percent lymphocytes:neutrophils. However, drug deliveredin PBTE alone at 30 mg/kg/day showed a slight decrease in WBCs after 2-3weeks of treatment. Animals given 40 mg/kg/day showed both anemia andleukopenia after several weeks of treatment when given A10 in PBTEalone. No effects on blood cell were observed when A10 was administeredin PBTE:D5W even after 100 days of treatment.

Effects of A10 on Body Weight

The effect of daily administration of A10 (20 mg/kg/day) on body weightwas examined over a 100 day period. A10 had an initial effect on weightgain compared to controls. However, after several weeks, the animalsgained weight at a rate similar to untreated or vehicle-treated animals.Over the treatment period, no mortality was observed. In addition therewere no effects on blood differentials and no observable effects onmajor organ histopathology, including heart, liver, lung, kidneys,spleen, long bone, stomach, mesenteric lymph node, small and largeintestine and pancreas.

Determination of LD₅₀

The lethal dose of A10 for 50% of animals (LD₅₀) was determined for bothathymic mice (BALB/c, nu/nu, female) and BALB/c mice (male and female)using a number of dosing regimens. As shown in Table XXV, the LD₅₀ ofA10 ranged from 83-145 mg/kg.

The effects of Dilantin® (phenytoin sodium for injection), ananti-convulsant agent, and Decadron® (dexamethasone sodium phosphate forinjection), an anti-inflammatory agent, on the LD₅₀ of A10 were alsodetermined (Table XIV). The LD₅₀ for A10 following pretreatment ofanimals with Decadron® or Dilantin® was 94 and 144 mg/kg, respectively.

                  TABLE XXV                                                       ______________________________________                                        Determination of A10 LD.sub.50 in mice                                                            Strain/  LD.sub.50                                        Dose/Regimen        sex      (mg/kg)                                          ______________________________________                                        single dose         athymic, 145                                                                  f                                                         every 4 days × 4                                                                            athymic, 75                                                                   f                                                         every 7 days x 4    athymic, 100                                                                  f                                                         single dose         BALB/c,  83                                                                   f                                                         single dose         BALB/c,  107                                                                  m                                                         single dose with Decadron ®                                                                   BALB/c,  94                                               pretreatment        f                                                         single dose with Dilantin ®                                                                   BALB/c,  144                                              pretreatment        f                                                         ______________________________________                                    

Table XXV. Decadron® was administered at 1.5 mg/kg/day for 7 days priorto administration of a single dose of A10. Dilantin® was administered at20 mg/kg/day for 7 days prior to administration of a single dose of A10.The LD₅₀ was calculated from a plot of % mortality versus dose (log M)using a four parameter logistic equation. f=female, m=male, n=5 animalsper group.

Example 14 In Vivo Inhibition of Tumor by a Mutated PDGF-R Receptor

This example illustrates the use of nucleic encoding a truncated PDGF-breceptor to inhibit in vivo tumor growth. C6 rat glioma cells wereinfected with retroviruses carrying a mutant gene for the human PDGF-breceptor. Seven G418-selected clones were screened for expression of thetruncated receptor by Western blotting with an antibody that recognizesthe extracellular domain of the human receptor but does not cross-reactwith the wild type rat receptor. Two clones, HiMut.1 and HiMut.2,express high levels of a protein with the predicted molecular weight forthe receptor lacking most of the intracellular region. Several clonesexpressed low levels of the truncated receptor; LoMut.1 was chosen forfurther experiments. Himut.1 expressed PDGF-R 8.3 fold higher thanLoMut.1. Himut.2 expressed PDGF-R 9.4 fold higher than LoMut.1. Theisolation and characterization of the clones containing the mutantreceptors were carried out as described below.

Cell culture. All culture media, fetal bovine serum (FBS) and chemicalswere purchased from Gibco BRL. C6 rat glioma cells were grown inHam's/F-10 medium supplemented with 5% fetal bovine serum and 2 mMglutamine. COS cells were cultured in 10% fetal bovine serum and 2 mMglutamine in Dulbecco's Modified Eagle's medium (DMEM).

Expression of mutant PDGF-b receptor. A stop codon was introduced bysite-directed mutagenesis into the gene for the human PDGF-b receptordirectly upstream from the first tyrosine kinase domain. The mutant genewas cloned into a vector under the control of the murine sarcoma viruslong terminal repeat (Muller, A. J., et al., Mol. Cell. Biol.11:1785-1792, 1991). Four mg each of this vector and a vector containingthe genes required for retroviral virus packaging (Muller, supra) werecotransfected into COS cells (2×10⁵ cells/60 mm plate) by calciumphosphate precipitation (Chen, C. A., and H. Okayama, BioTech.6:632-638, 1988). The cells were washed with PBS and refed the followingday and conditioned media collected on days 4-6 after transfection. C6cells (10⁵ cells/60 mm plate) were infected with dilutions of theconditioned media containing 6 mg/ml Polybrene (Sigma). Two days later,the cells were put into selection with 800 mg/ml G418 (Gibco) andcolonies picked when distinguishable. The vector control cells wereprepared by the same method but with a vector lacking a gene under theLTR.

Co-immunoprecipitation of wild type and truncated receptors. Vectorcontrol cells and cells expressing high levels of the mutant PDGF-breceptor (HiMut.1) were each seeded with 3×10⁵ cells/well on 6-wellplates. The following day, the media was changed for 0.5 ml 3% FBS in-cys -met DMEM (ICN) containing 100 mCi/ml Tran³⁵ S-label (ICN). Thecells were incubated at 37° C. for 16 hrs. They were washed twice withbinding buffer (0.1% BSA, 10 mg/ml CaCl₂.2H₂ O, 10 mg/ml MgSO₄.7H₂ O, 10mg/ml aprotonin and 0.2 mM PMSF in PBS), and 0.5 ml binding buffer or0.5 ml 20 ng/ml PDGF-BB (Collaborative Research Inc.) in binding bufferwas added to each well. The cells were incubated at 4° C. for 4 hrs,washed twice with PBS and lysed with 0.5 ml 1% Triton X-100 in HNTG (20mM HEPES (pH 7.5), 150 mM NaCl, Triton X-100, 10% glycerol, 10 mg/mleach of aprotonin, leupeptin and pespstatin, and 0.2 mM PMSF). PDGF-BBwas included in the lysis buffer of cells that had been treated withPDGF. The lysates were spun at 100,000×g for 30 min at 4° C. Thesupernatants were transferred to new tubes and precleared with ProteinA-agarose (Vector Laboratories). SDS was added to a final concentrationof 0.1% to 2 PDGF-treated samples for each cell line. Duplicate sampleswere immunoprecipitated with either an antibody that recognizes theC-terminus of the wild type rat receptor (UBI anti-PDGF-b receptor) orthe human mutant receptor (Genzeme anti-PDGF-b receptor). Rabbitanti-mouse IgG was used as a secondary antibody for the samplesincubated with the Genzyme anti-receptor. The complexes wereprecipitated with Protein A-agarose and washed 5 times with 0.1% TritonX-100 in HNTG. The proteins were separated by SDS-polyacrylamide gelelectrophoresis on 7.5% gels under reducing conditions. The gels werefixed, treated with Amplify (Amersham) and exposed to X-ray film for 3days.

Western blotting. Each cell line was plated in multiple wells at 5×10⁵cells/well on 6-well plates. The following day they were fed with 1% FBSin MCDB 105 (UCSF Cell Culture Facility) and incubated for 24 hrs in a0% CO₂ environment. PDGF-AA or -BB (Collaborative Research Inc.) wasadded to one well of each clone to the desired final concentration.After incubating for 7 min at room temperature, the cells were washedwith PBS and lysed with 50 mM Tris-HCl (pH 7.4), 1% nonidet P-40, 10%glycerol, 2 mM EDTA, 10 mM sodium pyrophosphate, 10 mg/ml each aprotininand leupeptin, 1 mM PMSF and 1 mM sodium orthovanadate. Equal volumes ofeach lysate were run on multiple 7.5% SDS polyacrylamide gels andtransferred to nitrocellulose (Schleicher & Schuell). The membranes wereblocked with 5% instant nonfat milk in Tris-buffered saline/0.05%Tween-20 (TBST-T). Duplicate membranes were incubated with eitherpolyclonal anti-phosphotyrosine 1:3000 or anti-PDGF-b receptor (UBI)1:1000 in blocking buffer. The secondary antibody was horseradishperoxidase-conjugated goat anti-rabbit IgG (Sigma) 1:1000. To detect thetruncated receptor, a monoclonal antibody against the extracellulardomain of the human PDGF-b receptor (Genzyme) diluted 1:500 wasutilized. The secondary antibody used was peroxidase-conjugated rabbitanti-mouse IgG (ICN) 1:1000. ECL (Amersham) was used for detection onall blots. Relative band areas were determined with a Molecular DevicesComputing Densitometer. Basal levels of phosphorylation were subtractedfrom each point.

Adherent growth of cell lines. To determine growth densities, each cellline was seeded with 10⁴ cells/well on 5 24-well plates with triplicatesamples in 1% or 5% FBS in Ham's/F-10 medium. The media was changedevery 3 days. Every 2 days, the cells on one plate were trypsinized andcounted on a Coulter counter. To determine cloning efficiencies, 100cells of each cell line were plated on three 10 cm plates in 1% or 5%FBS in Ham's/F-10 medium. The media was changed every 3 days for about12 days. The colonies were fixed, stained with methylene blue andscored.

Anchorage-independent growth of cell lines (soft agar assay). A baselayer was made in 35 mm plates with 0.8% SeaPlaque agarose (FMCBioProducts), 1% FBS 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPESand nonessential amino acids. Cells were suspended in 0.4% agarosecontaining the other ingredients listed above arid the desiredconcentration of PDGF-BB (Collaborative Research Labs). The suspensionwas plated on the base layer with 3000 cells/plate. They were incubatedfor 2 weeks in a humidified chamber with 5% CO₂. Colonies were scoredvisually or with an automated Omincon 3800™ tumor colony counter.

Growth of cell lines in nude mice. Cells were expanded in rollerbottles, trypsinized and resuspended in PBS. They were counted and thevolume adjusted to 3×10⁷ cells/ml. For each cell line, 4 to 8 athymicnude mice (Simonsen Labs) were injected subcutaneously with 100 ml(3×10⁶ cells). Tumor volumes were measured with calipers twice a weekfor 18 to 21 days.

Immunohistochemical staining of tumor sections. Tumors were resectedfrom the mice and frozen in OCT (Miles Lab). Five mm thick sections werecut and fixed with acetone. The sections were blocked with 10% normalgoat serum prior to incubation with 20 mg/ml biotinylated-anti-humanPDGF-b receptor (Genzyme antibody, biotinylated by Molecular Probes).Peroxidase-conjugated streptavidin (Caltag) 1:100 and diaminobenzidine(Sigma) with H₂ O₂ were used for detection. For a negative control, abiotinylated monoclonal antibody to an unrelated protein was used at thesame protein concentration as the anti-PDGF-b receptor. The counterstain was Harris hematoxylin (Anatech).

Using the retroviral expression system described above, a truncatedPDGF-b receptor was introduced into rat C6 glioma cells. InG418-resistant clones expressing the mutant receptor, PDGF-BB-inducedtyrosine phosphorylation of the wild type receptor was significantlyreduced. Furthermore, these cells grew to lower density and formedsmaller colonies in culture and in soft agar than the parental C6 cells.

Cells expressing the truncated receptor were significantly impaired intheir ability to grow in nude mice. After 21 days, the volumes of thetumors from HiMut.1 and HiMut.2 were only 12-16% of the size of thetumors from the parental cells. Tumors derived from the C6 parentalcells and vector control cells were essentially identical indicatingthat G418 selection of the vector control cells did not affect theirability to grow in nude mice. Tumors derived from HiMut.1 gave very darkimmunological staining in at least 10% of the cells. HiMut.2-derivedtumors were stained in 45-85% of the cells. The presence of thetruncated PDGF-b receptor was also confirmed by western blotting oflysed tumor sections. Thus, the truncated PDGF-b receptor was expressedin vivo for up to 21 days and it had to be present in at least 10% ofthe cells to be inhibitory. These studies demonstrate the usefulness ofdominant negative mutants of PDGF-R to inhibit growth of tumorscharacterized by inappropriate PDGF-R activity in vivo.

Example 15 Cellular Culture and In vitro Affects of Other Compounds

This example illustrates the effect of featured compounds, other thanA10 and B11, on tumor growth in cell culture or in vivo. PDGF-Ractivity, C6 SRB, and in vivo efficacy were measured using theprocedures described in the appendices above. The results are shown inTable XXVI and XXVII. These results are preliminary data. One skilled inthe art can improve the efficacy of the different compounds usingfactors known in the art such as different dosing regimens.

                                      TABLE XXVI                                  __________________________________________________________________________        U1242                                                                              C6 SRB   C6 in vivo                                                  Comp                                                                              KINASE                                                                             IC.sub.50                                                                          LD.sub.10                                                                         EFFICACY                                                    ound                                                                              IC.sub.50 (μM)                                                                  (μM)                                                                            (mg/kg)                                                                           (% at mg/kg)                                                                          p Value                                                                           Comments                                        __________________________________________________________________________    P10 3.0 (W)                                                                            12                                                                   J10 1 (W)     171 73% at 40   100% mortality                                      >50  12                   at d18                                          G13 1.5  >50  not 12% at 15   0% mortality                                                  tested                                                          G14 0.8 (W)                                                                            >50  not 36% at 15                                                                             0.285                                                                             0% mortality at                                               tested          d14                                             G21 6.0 (W)                                                                            >12  >200 no                                                                           no effect at 40                                                                           0% mortality                                        >50  >12  deaths                                                          G22 0.9 (W)                                                                            >6   25  no effect at 20                                                                           0% mortality                                        14   >6                   only one dose                                                                 tested in MTD                                                                 due to                                                                        solubility                                                                    problems                                        P16 0.8       >400 no                                                                           no effect at 60                                                                           0% mortality                                        >50  17   deaths                                                          F10 45 (W)                                                                             >12.5                                                                G24 0.6  1    >200 no                                                                           no effect at 20                                                                           0% mortality                                        5    2.2  deaths                                                                            no effect at 40                                                                           12.5% mortality                                                   no effect at 60                                                                           at d15                                                                        100% mortality                                                                at d10                                          G25 1.0  50                                                                   P13 1.2  >100                                                                 P17 0.8/3.0                                                                            12   >100 no         only one dose                                       >50       deaths          tested in MTD                                                                 due to                                                                        solubility                                                                    problems                                        H10 25 (W)    75  52% at 30                                                                             0.006                                                                             14.3% mortality                                     >50  13               0.12                                                                              at d17                                          H12 >250 (W)                                                                           19                                                                       25 (W)                                                                    H13 >50  20   >400 no                                                                           0% at 40                                                                              0.12                                                                              0% mortality                                        >50  10.5 deaths                                                                            41% at 100                                                                            0.12                                                                              0% mortality                                    E15 5.0 (W)                                                                            4.4  >400 no                                                                           66% at 15                                                                             0.019                                                                             0% mortality                                        10 (W)    deaths                                                                            58% at 50                                                                             0.1 12.5% mortality                                     >50           0% at 100   at d15                                                                        100% mortality                                                                at d8                                           P19      >50                                                                  P21      >25                                                                  J11 3.1  14                                                                   P21 93   >25                                                                  __________________________________________________________________________     W = done in Western assay                                                

                  TABLE XXVII                                                     ______________________________________                                                U1242                                                                         KINASE   C6 SRB   LD.sub.10                                           Compound                                                                              IC.sub.50 (μM)                                                                      IC.sub.50 (μM)                                                                      (mg/kg)  Comments                                   ______________________________________                                        A13              >>25     >200 no deaths                                                                         ˜30% inhibition                                                         at 25 μM                                P22              >>25              ˜10% inhibition                                                         at 25 μM                                P23              18                                                           P24              14                                                           G28     16.4     4.5                                                                  (scndry)                                                              G29     23.2     >13                                                                           killed                                                                        all cells                                                                     at >12.5                                                                      μM                                                        G30     32.5     2                                                            ______________________________________                                         W = done in Western assay                                                

Example 16 Combination Therapy

Studies were conducted to look at the effect on tumor growth when PDGF-Rinhibitors are used in combination with known cytotoxic drugs currentlyused to treat cancer. In separate experiments CALU-6 cells andMCF-7/Her2 cells were implanted subcutaneously in nude mice. In oneexperiment the mice were then treated with A10 alone, cisplatin alone ora combination of cisplatin and A10. A10 was given intraperitoneallytwice weekly at 5/mg/kg. Cisplatin was given in a single intraperitonealdose of 5/mg/kg on day two.

In the mice implanted with MCF-7/Her2 cells, the combination of A10 andcisplatin was better at inhibiting tumor growth than cisplatin alone orno treatment, but did not enhance tumor suppression compared to A10alone. However, in the mice implanted with CALU-6 cells, the combinationof A10 and cisplatin resulted in a significant suppression of tumorgrowth compared to A10 alone, cisplatin alone or no treatment.

In a second experiment with CALU-6 cells, mice were treated with A10,cisplatin or VP-16 alone (10/mg/kg on days 4, 7 and 10) or thecombination of cisplatin and VP-16 or A10, cisplatin and VP-16. Thecombination of A10, cisplatin and VP-16 was better at suppressing tumorgrowth than either drug alone or the combination of cisplatin and VP-16.

Example 17 Chemical Synthesis

Some of the compounds of this invention may be prepared by employingprocedures known in the literature starting from known compounds orreadily made intermediates. Quinoxalines compounds were prepared byeither 1) reacting 1,2 aromatic diamine with a-ketoaldehyde ora-diketone, or 2) an exchange reaction of a-bis thiosemicarbazones and a1,2-diamine in the presence of an acid catalyst. In the followingpreparations the aromatic diamine was obtained commercially or preparedas described in the example. In examples where the reaction solvent isnot specified, the reaction was carried out in ethanol-acetic acid. Somequinoxalines synthesized using this solvent were isolated as acidaddition complexes with one molecule acetic acid, band on elementalanalysis. Reactions in ethanol alone, followed by solvent evaporationand recrystallization, gave a cleaner product and higher yield.

Group 1 Compounds

A10

A10 can be prepared as in European Patent Application 0 013 376 A2.Alternatively A10 can be prepared as follows:

Step 1: Preparation of acetoacetic acid-(4-trifluoromethyl)aniline

A mixture of 4-trifluoromethylaniline (16.1 g, 0.1 mol),2,2,6-trimethyl-4H-1,3-dioxin-4-one (purity 95%; 14.97 g, 0.1 mol), andxylene (20 ml) was heated to reflux for 30 minutes in a bath preheatedto 150° C. The resulting dark solution was cooled to room temperature tocrystalize the product. The crystals were filtered and collected. Morematerial was obtained from mother liquors (the solution remaining afterthe initial crystallization and filtration). The yield of crude anilidewas 17.75 g (72%), the anilide had a melting point of 153-154° C.

Step 2: Preparation of2-Ethoxymethyleneacetoacetyl-(4-trifluoromethyl)aniline

Acetoacetyl-(4-trifluoromethyl)aniline (14.11 g., 57.6 mmol),triethoxymethane (9.43 g, 63.4 mmol), and acetic anhydride (16.30 ml,173 mmol) were mixed together and heated to reflux for 90 minutes. Theresulting dark solution was evaporated to dryness. The residue wasresuspended in benzene/isooctane and the product was crystallized. Morematerial was obtained from mother liquors. Yield of pure product was11.93 g (72%), mp. 120-122° C.

Step 3: Preparation of A10

2-Ethoxymethyleneacetoacetyl-(4-trifluoromethyl)aniline (3.01 g, 10.4mmol) in ethanol (6 ml) was slowly added to an ice cooled solution ofhydroxylamine hydrochloride (0.77 g, 11.0 mmol) in 2 M NaOH (5.5 ml).The mixture was heated to reflux for 1 hour, cooled to room temperatureand evaporated to dryness. The residue was resuspended, and distributedbetween ethyl acetate and water. The organic layer was separated,extracted with water, dried by sodium sulfate and the solvent wasevaporated. The residue was resuspended in toluene and crystallized toyield a solid residue (2.45 g, 87%) of A10 having a melting point of166-167° C.

A11

Preparation of 5-Methyl-isoxasole-4-carboxylicacid-(3-trifluoromethyl)-anilide was carried out in three steps

a) Preparation of Acetoacetic acid--(3-trifluoromethyl)-anilide

In a 25 ml round bottom flask equipped with Claisen distillation headand magnetic stirrer 4 g (18.6 mM) of α,α, α-Trifluoro-p-toluidine, 3.71g (24.8 mM, 1 equiv.) 95% 2,2,6-trimethyl-4H-1,3-dioxin-4-one, 112 mldiethanolamine and 12.4 ml xylene was combined. The temperature of themixture was raised to 110° C., and the mixture was stirred at thistemperature for 6 hours while acetone was distilled off from the system.The progression of the reaction was monitored by TLC (plate MerckKieselgel 60 F₂₅₄ eluent: Petroleum ether (90-110° C. fraction):acetone2:1) visualization with 5% PMA in EtOH).

After 6 hours the xylene was distilled off at 20 Hgmm and the residuewas purified with medium pressure (2 atm) liquid chromatography usingSilica gel 60 as fixed phase and petroleum ether (90-110° C.fraction):acetone 2:1 as eluent.

The product fractions at R_(f) =0.3 were collected--using the TLC systemused for the monitoring of the reaction--and after stripping thesolvent, 3.82 g of Acetoacetic acid--(3-trifluoromethyl)-anilide wasisolated. Melting point: 91-92° C.

¹ H-NMR (ppm, acetone-d6) ArH 7.36-7.87 4H (m) NH 9.41 1H (s) CH₂ 3.622H (s) CH₃ 2.64 3H (s)

b) Preparation of 2-propenamide,2-acetyl-3-ethoxy-N-[(trifluoromethyl)phenyl]

In a dried (120° C., 30 min) 50 ml round bottom flask equipped withmagnetic stirrer, thermometer, rubber septum and Argon balloon on a Tstopcock, 1.0 g (4.1 mM) Acetoacetic acid-(3-trifluoromethyl)-anilide,0.85 g (5.7 mM) orthoformic acid triethylesther 1.37 g acetic anhydrideand 450 mg dry zinc chloride was combined. The mixture was stirred inargon atmosphere at 60° C. for 30 minutes. The progress of the reactionwas monitored by TLC (plate Merck Kieselgel 60 F₂₅₄, eluent: Petroleumether (90-110° C. fraction):acetone 5:1. If the reaction was notcomplete after 30 minutes, a further 0.85 g (5.7 mM) of orthoformic acidtriethylesther was added.

The reaction mixture gradually turned brown. After the completion of thereaction, the reaction mixture was stripped in vacuo, the residue wasdissolved in ethyl acetate, which was washed with water, the organicphase was dried with magnesium sulfate then filtered and the ethylacetate was distilled off in vacuo. The 1.16 g crude product waspurified with medium pressure (2 atm) liquid chromatography using Silicagel 60 as fixed phase and petroleum ether (90-110° C. fraction):acetone5:1 as eluent.

The product fractions at R_(f) =0.231 were collected--using the TLCsystem used for the monitoring of the reaction--and after stripping thesolvent, 0.46 g of 2-propenamide,2-acetyl-3-ethoxy-N-[(trifluoromethyl)phenyl] was isolated. Meltingpoint: 100.5° C.

¹ H-NMR (ppm, acetone-d6) ARH 7.29-8.00 4H (m) NH 11.25 1H (s) CH═ 8.461H (s) EtCH₂ 4.37 2H (s) EtCH₃ 1.45 3H (s) AcMe 2.48 3H (s)

c) Preparation of 5-Methyl-isoxasole-4-carboxylicacid-(3-trifluoromethyl)-anilide

In a 25 ml round bottom flask equipped with magnetic stirrer andthermometer 0.11 g (1.58 mM) of hydroxylamine hydrochloride wasdissolved in 0.5 ml of water and to this solution 64 mg (1.6 mM) sodiumhydroxide was added in 0. 5 ml of water. To this solution 2.2 ml ofmethanol was added and at room temperature 0.44 g (1.5 mM)2-propenamide, 2-acetyl-3-ethoxy-N-[(trifluoromethyl)phenyl] was added.The progress of the reaction was monitored by TLC (plate Merck Kieselgel60 F₂₅₄ eluent: Petroleum ether (90-110° C. fraction) :acetone 4:1,visualization with 5% PMA in ETOH. After completion of the reaction, thereaction mixture was concentrated in vacuo and the residue was dissolvedin ethyl acetate. The organic phase was washed with water, dried withmagnesium sulfate, filtered and concentrated. The obtained 0.51 g crudeproduct was purified with medium pressure (2 atm) liquid chromatographyusing Silica gel 60 as fixed phase and petroleum ether (90-110° C.fraction) :acetone 4:1 as eluent. The product fractions at R_(f) =0.162were collected--using the TLC system used for the monitoring of thereaction--and after the stripping of the solvent, 0.22 g of5-Methyl-isoxasoile-4-carboxylic acid-(3-trifluoromethyl)-anilide wasisolated. Melting point: 115-120° C.

¹ H-NMR (ppm, acetone-d6) ARH+NH 7.42-7.88 4H (m) CH═ 8.50 1H (s) CH8.50 1H (s)

A12

A12 can be prepared as described by Patterson et al., J. Med.

Chem. 35:507-510 (1992).

A13: N-trifluoromethlyphenyl 3,5-dimethylisoxazole-4-carboxamide

A mixture of 320 mg of 3,5-dimethylisoxazole-4-carbonylchloride and 1 mlof 4-trifluoromethlyaniline in 2 ml of dichloromethane was stirred atroom temperature overnight. The mixture was then worked up with ethylacetate and water. The crude was crystallized with ethanol and water toprovide 330 mg of N-trifluoromethlyphenyl3,5-dimethylisoxazole-4-carboxamide.

Group 2 Compounds

B10

B10 was synthesized in two steps.

a) Synthesis of cyanoacetyl-(4-nitro)anilide

1.38 g (10 mmol) 4-nitroaniline was dissolved in 30 ml of absolutepyridine, then cooled to -30° C., 0.43 ml (5 mmol) phosphorustrichloride was added dropwise with continuous stirring to avoidincreasing the temperature above -20° C. After 0.5 hour 0.85 gcyanoacetic acid was added, and the solution was stirred for 0.5 hour at-20° C. then 12 hours at room temperature.

The solvent was evaporated in vacuum. The residue was covered with 1NHCl and extracted with ethylacetate. The ethylacetate solution was driedover Na₂ SO₄, filtered and evaporated. The residue was triturated withether filtered and dried in vacuo. 1.70 g (83%) product yield wasobtained. The product had the following characteristics:

Melting point: 81-83° C.

R_(F) : 0.95 (hexane-EtOAc; 1:1)

b. Acetylation of cyanoacetyl-(4-nitro)anilide

0.82 g (4 mmol) cyanoacetyl 4-nitroanilide was dissolved in 2 mlabsolute pyridine, followed by the addition of 20 mg 4-aminopyridinecatalyst and 0.50 ml tetramethylguanidine. The. reaction mixture wasstirred while the nitroanilide dissolved. 5 ml of acetic anhydride wasthen dropped in at 0° C.

After 2 days the pyridine was evaporated, ethylacetate was added and theorganic layer was extracted with 5 % NaHCC)₃ solution, in 1N HCl, andwater. The organic layer was dried over Na₂ SO₄ and evaporated. Theresidue was crystallized from ether and 210 mg (21% yield) of productwas obtained. The product had the following characteristics:

Rf=0.35 (EtoAc)

Melting point: 260° C.

B11

B11 was synthesized using two different methods: method A and method b.

Method A

A mixture of 27 grams of A10 in 150 ml of ethanol was combined with 16.2grams of 1,8-diazabicyclo[5.4.0]undec-7-ene. The reaction mixture wasthen stirred at room temperature for 30 minutes and ethanol wasevaporated off. The resulting solid was suspended in 500 ml of ethylether and combined with 200 ml of 0.6N hydrochoric acid solution. Allthe solids was suction filtered, washed with 200 ml ethanol and suctiondried to provide 25 grams product.

Method B

2-Butenamide, 2-cyano-3-hydroxy-N-[(4-trifluoro-methyl)phenyl](C₁₂ H₁₉F₃ NO₃) was prepared in two steps as follows

a) Preparation of Cyanoacet-(4-trifluoromethyl)-anilide

A mixture of 3 g (18.6 mM) a,a,a-Trifluoro-p-toluidine and 3.37 g (29.8mM, 1.6 equivalent) cyanoacetic acid ethyl ester in a 50 ml flask,equipped with magnetic stirrer, thermometer and nitrogen vent, wasstirred on a 180° C. oil bath for 5 hours. The progress of the reactionwas monitored by TLC (plate Merck Kieselgel E60 F₂₅₄, eluent: Petroleumether (90-110° C. fraction):acetone 1:1. The reaction mixture waspurified with medium pressure (2 atm) liquid chromatography using Silicagel 60 as fixed phase and petroleum ether (90-110° C. fraction):acetone1:1 as eluent.

The product fractions were collected--using the TLC system used for themonitoring of the reaction--and after stripping the solvent, 2.11 g ofCyanoacet-(4-trifluoromethyl)-anilide was isolated. Melting point:192-194° C.

1 H-NMR (ppm, Acetone-d6) ArH 7.68-7.85 4H (dd) NH 9.84 H (s) CH₂ 3.902H (s)

b) Preparation of 2-Butenamide,2-cyano-3-hydroxy-N-[(4-trifluoromethyl)phenyl]

In a 50 ml round bottom flask equipped with magnetic stirrer,thermometer, a rubber septum and a T stopcock (with vacuum and Argonblanket balloon joints), 1.78 g (0.89 g, 37.1 mM) 50% NaH oilydispersion was suspended in 4 ml dry (from P₂ O₅) acetonitrile. Thesuspension was cooled to 10° C. and while stirring at this temperature2.72 g (11.9 mM) Cyanoacet-(4-trifluoromethyl)-anilide was addeddissolved in 25 ml dry (from LiAlH₄) tetrahydrofuran in 10 minutes. Thereaction mixture turned yellow, and was cooled down after the additionto -10° C., and then 1.05 g (13.11 mM, 1.1 equiv.) Acetylchloride wasadded in 20 minutes. During the addition, the temperature of thereaction mixture can not be higher than -5° C. The progress of thereaction was monitored by TLC (plate Merck Kieselgel 60 F₂₅₄, eluent:Petroleum ether (90-110° C. fraction):acetone 1:1.

When the reaction was complete, the reaction mixture was stirred at 0°C. for 30 minutes, at 35° C. for 30 minutes and at 65° C. for another 30minutes, then the reaction mixture was stripped in vacuo. The residuewas dissolved in 30 ml distilled water, charcoaled at 80° C. andfiltered. The resulting pale yellow filtrate was acidified with a 10%hydrochloric acid solution, the precipitated crystals were filtered,washed with water and dried. The crude crystals (2.51 g) were purifiedwith medium pressure (2 atm) liquid chromatography using Silica gel 60as fixed phase and petroleum ether (90-110° C. fraction):acetone 1:1 aseluent.

The product fractions at Rf=0.138 were collected--using the TLC systemused for the monitoring of the reaction--and after stripping thesolvent, 1.99 g of 2-Butenamide,2-cyano-3-hydroxy-N-[(4-trifluoromethyl)phenyl] was isolated.

TLC petroleum ether (90-110° C.)/acetone 1/1=0.138.

1 H-NMR (ppm, DMSO-d6) ArH 7.65-7.78 4H (dd) NH 10.85 1H (s) CH₃ 2.26 3H(s) OH 6.39 1H (s)

B12

Preparation of 2-Propenamide,2-cyano-3-hydroxy-3-(4-fluorophenyl)-N-[(4-trifluoromethyl)phenyl] (C₁₇H₁₀ F₄ N₂ O₂ MW:350.3) was carried out as follows:

a) Preparation of Cyanoacet-(4-trifluoromethyl)-anilide

A mixture of 3g (18.6 mM) a,a,a-Trifluoro-p-toluidine and 3.37 g (29.8mM, 1.6 equivalent) cyanoacetic acid ethyl ester in a 50 ml flask,equipped with a magnetic stirrer, thermometer and nitrogen vent, wasstirred on a 180° C. oil bath for 5 hours. The progress of the reactionwas monitored by TLC (plate Merck Kieselgel 60 F₂₅₄, eluent: Petroleumether (90-110° C. fraction) :acetone 1:1. The reaction mixture waspurified with medium pressure (2 atm) liquid chromatography using Silicagel 60 as fixed phase and petroleum ether (90-110° C. fraction):acetone1:1 as eluent.

The product fractions were collected and after the stripping of thesolvent, 2.11 g of cyanoacet-(4-trifluoromethyl)-anilide was isolated.The product had the following characteristics: Melting point: 192-194°C.

¹ H-NMR (ppm, acetone-d6) ArH 7.68-7.85, 4H (dd) NH 9.84 1H (s) CH₂ 3.902H (s)

b) Preparation of 2-Proponamide,2-cyano-3-hydroxy-3-(4-fluorophenyl)-N-[(4-trifluoromethyl)phenyl]

In a 50 ml round bottomed flask equipped with magnetic stirrer,thermometer, a rubber septum and a T stopcock (with vacuum and Argonblanket balloon joints) 0.55 g (0.275 g, 11.4 mM) 50% NaH oilydispersion was suspended in 1 ml dry (from P₂ O₅) acetonitrile. Thesuspension was cooled to 10° C. and under stirring at this temperature 1g (4.4 mM) cyanoacet-(4-trifluoromethyl)-anilide, dissolved in 10 ml dry(from LiAlH₄) tetrahydrofuran, was added in 10 minutes. The reactionmixture was then cooled down to -10° C. and at this temperature 0.77 g(4.8 mM, 1.1 equiv.) 4-fluorobenzylchloride was added in 20 minutes.During the addition the temperature of the reaction mixture was notallowed to go higher than -5° C. The progress of the reaction wasmonitored by TLC (plate Merck Kieselgel 60 F₂₅₄, eluent: Petroleum ether(90-110° C. fraction):acetone 1:1.

The reaction mixture was then stirred at 0° C. for 30 minutes, at 35° C.for 30 minutes and at 65° C. for another 30 minutes, then the reactionmixture was stripped in vacuo. The residue was dissolved in 30 mldistilled water, charcoaled at 80° C. and filtered. The resulting paleyellow filtrate was acidified with 10% hydrochloric acid solution, theprecipitated crystals were filtered, washed with water and dried. Crudecrystals (2.47 g) were purified with medium pressure (2 atm) liquidchromatography using Silica gel 60 as fixed phase and petroleum ether(90-110° C. fraction):acetone 1:1 as eluent.

The product fractions at Rf=0.216 were collected and after the strippingof the solvent, 1.05 g of 2-Propenamide,2-cyano-3-hydroxy-3-(4-fluorophenyl)-N-[(4-trifluoromethyl)phenyl] wasisolated. The product had the following characteristics:

Melting point: 195° C. (dec).

¹ H-NMR (ppm, acetone-d6) ArH 7.36-8.13 8H (m) NH 9.5 1H (s) OH 16.5 1H(s)

B13

Preparation of 2-Propenamide,2-cyano-3-hydroxy-3-cyclohexyl-N-[(4-trifluoromethyl)phenyl] (C₁₇ H₁₇ F₃N₂ O₂ MW: 338.3) was carried out as described for B12 substituting 0.71g (4.8 mM, 1.1 equiv.) cyclohexylacetyl-chloride for4-fluorobenzoylchloride. The resulting product had the followingcharacteristics:

TLC: R_(f) =0.265 (petroleum ether (90-110° C.):acetone, 1:1) Meltingpoint: 212° C. (dec.)

B14

Preparation of2-cyano-3-hydroxy-3-(2,2,3,3-tetramethylcyclopropyl)-propanol-4-(trifluoromethyl)anilidewas carried out as described for B12 above using2,2,3,4-tetramethylcylopropylcarboxyl chloride for4-fluorobenzoylchloride.

B15

Preparation of 2-Propenamide,2-cyano-3-hydroxy-3-(pentafluorophenyl)-N-[(4-trifluoromethyl)phenyl]was carried out as described for B12 substituting 1.5 g (6.51 mM, 1.1equiv.) pentafluorobenzoylchloride for 4-fluorobenzoylchloride. Theresulting product had the following characteristics:

TLC: R_(f) =0.360 (petroleum ether (90-110° C.):acetone, 1:1)

Melting point: 157-158° C.

B16

Preparation of 2-Propenamide,2-cyano-3-hydroxy-3-((3-phenoxy)phenyl)-N-[(4-trifluoromethyl)phenyl](C.sub.23H₁₅ F₃ N₂ O₃ MW: 424.4) was carried out as described for B12substituting 1.65 g (4.8 mM, 1.1 equiv) 3-phenoxybenzoyl chloride for4-fluorobenzoylchloride. The resulting product had the followingcharacteristics:

TLC: R_(f) =0.300 (petroleum ether (90-110° C.):acetone, 1:1)

Melting point: 197-198° C.

B17

Preparation of 2-Butenamide,2-cyano-3-hydroxy-4-phenyl-N-[(4-trifluoromethyl)phenyl] was carried outas described for B12 substituting 0.68 g (4.8 mM, 1.1 equiv.)phenyl-acetyl chloride for 4-fluorobenzoylchloride. The resultingproduct had the following characteristics:

TLC: R_(f) =0.165 (petroleum ether (90-110° C.):acetone, 1:1)

Melting point: 156-158° C.

B18

Preparation of 2-Hexeneamide,2-cyano-3-hydroxy-5-methyl-N-[(4-trifluoromethyl)phenyl] (C₁₅ H₁₅ F₃ N₂O₂ MW: 312.3) was carried out as described for B12 substituting 0.58 g(4.8 mM, 1.1 equiv.) isovalerylchloride for 4-fluorobenzoylchloride. Theresulting product had the following characteristics:

TLC: R_(f) =0.323 (petroleum ether (90-110° C.):acetone, 1:1)

Melting point: 161-163° C.

B19

Preparation of 2-Butenamide,2-cyano-3-hydroxy-4,4-diphenyl-N-[(4-trifluoromethyl)phenyl] (C₂₄ H₁₇ P₃N₂ O₂ MW: 422.4) was carried out as described for B12 substituting 1.12g (4.8 mM, 1.1 equiv.) diphenylacetyl-chloride for4-fluorobenzoylchloride. The resulting product had the followingcharacteristics:

TLC: R_(f) =0.354 (petroleum ether (90-110° C.):acetone, 1:1)

Melting point: 195-202° C.

Group 3 Compounds

C10

340 mg (1.5 mM) 1-phenyl-3-amino-4-cyano-5-cyanomethyl-2-pyrazole, 210mg (1.5 mM) 3,4-dihydroxy benzaldehyde and 4 drops of piperidine in 30ml ethanol were refluxed for 6 hours. Cooling and filtering gave 145 mgyellow solid. Evaporation of the solvent and trituration with CH₂ Cl₂-acetone gave another 145 mg yellow solid (56% yield). The product had amelting point of 147° C.

NMR acetone d6 d-7.87 (1H,S, Vinyl), 7.68 (1H,d, J=2.2 Hz, H₂) 7.66-7.45(5H,m, Ph), 7.28 (1H,dd, J=8.3.2.2 Hz, H₆). 6.92 (1H,d,J=8.3 Hz, H₅).

C11

C11 was synthesized using a two step procedure.

a. Synthesis of 3-amino-4-cyano-5-cyanomethyl-2 pyrozole

2.2 g malononitrile dimer and 0.9 ml N₂ H₄ in 20 ml of water were heatedfor 15 minutes at 100° C. Cooling and filtering gave 1.5 g (61% yield)of a white solid having a melting point of 187° C. (NMR acetone d₆ δ3.88(s).) (Cf. Carboni et al., J. Am. Chem. Soc. 80:2838 (1958), reportingm.p. 197° C.)

b. Condensation with dihydroxybenzaldehyde

To 0.28 g (2 mM) 3,4-dihydroxybenzaldehyde and 1.33 g (2.2 mM) of3-amino-4-cyano-5-cyanomethyl-2 pyrozole in 20 ml ethanol were addedthree drops piperidine and the reaction was refluxed 3 hours. Cooling,filtering and washing with ethanol gave 1.3 g (56% yield) of a yellowsolid having a melting point of 300° C.

C13

0.7 g, 3 mM, 3,5 di-t-butly-4-hydroxyaldehyde 0.46 g, 3.1 mM, 3-amino 4cyano 5-cyanomethyl pyrazole (prepared according to Carboni et al., J.Chem. Soc., 80:2838, 1958) and 40 mg β-alanine were refluxed 15 hours.Cooling and filtering gave 0.5 g, 46% yield, yellow solid, mp. 255° C.NMR CDCl₃ δ7.92(1H,S,vinyl), 7.80(2H,S), 5.76(1H,S,OH),3.75(2H,br,S,NH2), 1.48(18H,S). MS-364(M⁺ 1,28), 363(M⁺, 100%),348(M-CH₃,58) 292(M-56-CH₃, 31), 147(41), m/e.

Group 4 Compounds

D11

435 mg (3 mM) 3-formyl indole, 300 mg (4.5 mM) 2-thiocarboxamidoacetonitrile and 20 mg β-alanine in 30 ml ethanol were refluxed for sixhours. Cooling and filtering gave 0.47 g (81% yield of a yellow solidhaving a melting point of 238° C.).

D12

This was synthesized as for D11 except 1,1,4-tricyano-2-amino-1-propenewas used instead of the acetonitrile derivative. The final product had amelting point of 293° C.

D13

This was synthesized as for D11 except 2-carboxamidoacetonitrile wasused instead of the acetonitrile derivative. The final product had amelting point of 242° C.

D14

0.29 g (2 mM) 3-formyl indole, 0.29 g (2 mM),3-amino-4-cyano-5-cyanomethyl-2-pyrazole and 20 mg β-alanine in 30 ml ofethanol were refluxed 4 hours. Cooling and filtering gave 0.34 g (62%yield) of yellow solid having a melting point of 281° C.

NMR acetone d₆ 8.52 (1H,S, vinyl), 8.42 (1H,S,H₂), 7.79 (1H,m), 7.57(1H,m), 7.27 (2H,m), 6.17 (1H, br.S, NH). MS-274 (M+, 100%), 219(14),91(35), m/e.

D15

0.3 g (1.3 mM) 3-amino-4-cyano-5-cyanomethyl-2-pyrazole, 0.2 g (1.36 mM)of 1-(3-dimethylaminopropyl)-3-formyl indole and 20 mg β-alanine in 20ml ethanol were refluxed 4 hours. Evaporation, trituration with benzeneand filtering gave 0.4 g of yellow solid (94% yield) containing 10%3-amino-4-cyano-5-cyanomethyl-2-pyrazole. 0.4 g was chromatographed onsilica gel (70-220 mesh) eluting with 85:15 methylene chloride:methanolto give 0.12 g of a bright yellow solid having a melting point of 250°C.

NMR acetone d₆ δ8.45(1H₁ S₁ vinyl), 8.37(1H₁ S₁ H₂), 7.78(1H₁ m),7.60(1H₁ m). 7.28(2H₁ m), 4.47(2H₁ t₁ J=6.8 Hz), 2.29(2H₁ t₁ J=6.8 Hz),2.24(6H,S,N-(CH₃)₂). MS-360(M+1, 8%), 359(M+,31), 289(100), 261(15),144(6), m/e.

D16

0.4 g (1.7 mM) 3-amino-4-cyano-5-cyanomethyl-2-pyrazole, 0.3 g (1.73 mM)1-oxo-1-(3,4-dihyroxyphenyl)-2-cyanothane Hand 20 mg β-alanine in 20 mlethanol were refluxed 5 hours. Cooling and filtering gave 0.1 g of abrown solid. Preparative chromatography gave 20 mg (3% yield) of anorange solid having a melting point of 115° C.

NMR acetone d₆ δ8.72(1H, S, Vinyl), 8.52(1H₁ S₁ H2), 7.90(1H₁ m)7.73(1H,m), 7.40(4H,m), 7.0(1H,d,J=8.2 Hz, H₅). 4.57(2H,t,J=7.2 Hz),2.46(2H,t,J=7.2 Hz), 2.34(6H,S,N(CH₃)₂), 2.17(2H, quintet, J=7.2 Hz).

D17

0.4 g (2 mM) 3-formyl indole, 0.36 g1-oxo-1-(3,4-dihydroxyphenyl)-2-cyanothane and 3 drops of piperidine, in25 ml ethanol, were refluxed 6 hours. Workup and trituration withbenzene gave 0.36 g of a yellow solid having a melting point of 225° C.

NMR acetone d₆ 8.77(1H,S), 7.90(1H,m), 7.70(1H,m), 7.40(4H,m),7.0(2H,t,J=6.7 Hz), 4.92(2H,t,J=6.8 Hz), 3.26(2H,t,J=6.8 Hz).

D18

0.29 g, 2 mM, 3-formyl indole, 0.29, 2 mM,3-amino-4-cyano-5-cyanomethyl-2-pyrazole, and 20 mg β-alanine in 30 mlethanol were refluxed 4 hours. Cooling and filtering gave 0.34 g, 62%yield, yellow solid, mp. 281° C.

NMR acetone d₆ δ8.52 (1H,S, Vinyl), 8.42 (1H,S,H₂), 7.79 (1H,m), 7.75(1H,m), 7.27 (2H,m), 6.17 (1H, Br.S, NH), MS-274 (M⁺, 100%), 219(14),91(35), m/e.

D20

80 mg. 0.55 mM, 3-formyl indole, 130 mg, 0.6 mM,3-amino-4-cyano-5-cyanomethyl-1-phenylpyrazole and 2 drops of piperidinein 10 ml ethanol were refluxed 8 hours. Cooking and filtering gave 120mg, 62% yield, yellow-green solid, mp. 258° C.

NMR acetone d₆ δ8.56(1H,S), 8.52(1H,S), 7.84(1H,m, 7.60-7.25(8H,m).

Group 5

Group 5 compounds were prepared in three steps.

a) Preparation of N-aryl-oxamic acid esters (═Ethyl-oxalyl anilides)

0.025 mol (3.4 ml) diethyl-oxalate and 0.1 mol of the appropriateaniline were mixed together and refluxed at 190° C. for 15 minutes. Theresulting solution was cooled and left overnight to crystallize theproduct. The crystals were filtered, washed with ethanol and extractedwith hot ethanol. The insoluble material was filtered off and thesolution put in the refrigerator. The resulting crystals were filteredand dried.

                  TABLE XXVIII                                                    ______________________________________                                                                                 Yield                                No.   Subs.     MP ° C.                                                                          Formula: MW    [%]                                  ______________________________________                                        1a    4-N(CH.sub.3).sub.2                                                                     116-118   C.sub.12 H.sub.16 N.sub.2 O.sub.3                                                      236.27                                                                              75                                   1b    3-OH      184-185   C.sub.10 H.sub.11 NO.sub.4                                                             209.20                                                                              86                                   1c    2-OCH.sub.3                                                                             81-82     C.sub.11 H.sub.13 NO.sub.4                                                             223.23                                                                              60                                   1d    2-OC.sub.2 H.sub.5                                                                      74-76     C.sub.12 H.sub.15 NO.sub.4                                                             237.26                                                                              64                                   1e    3-NO.sub.2                                                                              93-96     C.sub.10 H.sub.10 N.sub.2 O.sub.5                                                      238.20                                                                              53                                   ______________________________________                                    

b) Preparation of N-aryl-oxamic acid hydrazides (N-aryl-oxamoylhydrazides)

0.05 mol of the appropriate N-aryl-oxamic acid ester (1a . . . 1e) wasdissolved in 200 ml of ethanol and slowly added to a well-stirredsolution of 7.5 ml (˜0.15 mol) hydrazine hydrate in 50 ml ethanol. Themixture was left at room temperature for 48 hours. The resultingheterogeneous solution was refluxed for 15 minutes and filtered the hotsolution. After cooling to room temperature the precipitated substancewas filtered washed with ethanol and dried.

                  TABLE XXIX                                                      ______________________________________                                                                                 Yield                                No.   Subs.     MP ° C.                                                                          Formula: MW    [%]                                  ______________________________________                                        2a    4-N(CH.sub.3).sub.2                                                                     228-232   C.sub.10 H.sub.14 N.sub.4 O.sub.2                                                      222.25                                                                              83                                   2b    3-OH      200-202   C.sub.8 H.sub.9 N.sub.3 O.sub.3                                                        195.18                                                                              72                                   2c    2-OCH.sub.3                                                                             165-167   C.sub.9 H.sub.11 N.sub.3 O.sub.3                                                       209.21                                                                              67                                   2d    2-OC.sub.2 H.sub.5                                                                      152-154   C.sub.10 H.sub.13 N.sub.3 O.sub.3                                                      223.23                                                                              58                                   2e    3-NO.sub.2                                                                              231-234   C.sub.8 H.sub.8 N.sub.4 O.sub.4                                                        224.18                                                                              49                                   ______________________________________                                    

c) Preparation of N-aryl-oxamoyl hydrazones

E10

0.001 mol (0.222 g) of N-(4-dimethylamino)-phenyl-oxamoyl hydrazide (2a)was dissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.138 g) of 3,4-dihydroxy benzaldehyde for 20-25 minutes in thepresence of a catalytic amount of sodium acetate. The mixture thencooled and left overnight at room temperature. The separated crystalswere filtered off and washed with acetic acid and water.

Yield of pure product was 0.23 g (68%) m.p. 253° C. (C₁₇ H₁₈ N₄ O₄,MW:342.36)

Elemental Analysis [%]: Found C, 59.51: H, 5.28; N, 16.25 Calculated C,59.64; H, 5.30; N, 16.37.

E11

0.001 mol (0.195 g) of N-3-hydroxy-phenyloxamoyl hydrazide (2b) wasdissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.138 g) of 3,4-dihydroxy benzaldehyde for 20-25 minutes in thepresence of a catalytic amount of sodium acetate. The mixture was thencooled and left overnight at room temperature. The separated crystalswere filtered off and washed with acetic acid and water.

Yield of pure product was 0.142 g (45%) m.p. >260° C. (C₁₅ H₁₃ N₃ O₅,MW:315.29).

Elemental Analysis [%]: Found C, 57.06; H, 4.10; N, 13.20. Calculated C,57.14; H, 4.16, N, 13.33.

E12

0.001 mol (0.195 g) of N-3-hydroxyphenyl-oxamoyl hydrazide (2b) wasdissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.122 g) of 2-hydroxy-benzaldehyde for 20-25 minutes in the presence ofa catalytic amount of sodium acetate. The mixture was then cooled andleft overnight at room temperature. The separated crystals were filteredoff and washed with acetic acid and water.

Yield of pure product was 0.224 g (75%) m.p. 264-266° C. (C₁₅ H₁₃ N₃ O₄,MW:299.29)

Elemental Analysis [%]: Found C, 60.11; H, 4.40; N, 13.76. Calculated C,60.20; H, 4.38; N, 14.04.

E13

0.001 mol (0.21 g) of N-2-methoxyphenyl-oxamoyl hydrazide (2c) wasdissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.138 g) of 3,4-dihydroxy-benzaldehyde for 20-25 minutes in thepresence of a catalytic amount of sodium acetate. The mixture was thencooled and left overnight at room temperature. The separated crystalswere filtered off and washed with acetic acid and water.

Yield of pure product was 0.21 g (64%) m.p. 232-238° C. (C₁₆ H₁₅ N₃ O₅,MW:329.31). Elemental Analysis [%]: Found C, 60.01; H, 4.51; N, 12.59.Calculated C, 58.36; H, 4.59; N, 12.72.

E14

0.001 mol (0.22 g) of N-2-ethoxyphenyl-oxamoyl hydrazide (2d) wasdissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.138 g) of 3,4-dihydroxy-benzaldehyde for 20-25 minutes in thepresence of a catalytic amount of sodium acetate. The mixture was thencooled and left overnight at room temperature. The separated crystalswere filtered off and washed with acetic acid and water.

Yield of pure product was 0.15 g (44%) m.p. 208-214° C. (C₁₇ H₁₇ N₃ O₅,MW:343.34). Elemental Analysis [%]: Found C, 59.78; H, 4.81; N, 12.10.Calculated C, 59.47; H, 4.99; N, 12.24.

E15

0.001 mol (0.22 g) of N-3-nitrophenyl-oxamoyl hydrazide (2e) wasdissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.138 g) of 3,4-dihydroxy-benzaldehyde for 20-25 minutes in thepresence of a catalytic amount of sodium acetate. The mixture was thencooled and left overnight at room temperature. The separated crystalswere filtered off and washed with acetic acid and water.

Yield of pure product was 0.19 g (56%) m.p. >260° C. (C₁₅ H₁₂ N₄ O₆,MW:344.286). Elemental Analysis [%]: Found C, 52.08; H, 3.47; N, 16.10.Calculated C, 52.32; H, 3.51; N, 16.27.

E16

0.001 mol (0.22 g) of N-3-nitrophenyl-oxamoyl hydrazide (2e) wasdissolved in 5 ml acetic acid and heated at 100° C. with 0.001 mol(0.122 g) of 4-hydroxy-benzaldehyde for 20-25 minutes in the presence ofa catalytic amount of sodium acetate. The mixture was then cooled andleft overnight at room temperature. The separated crystals were filteredoff and washed with acetic acid and water.

Yield of pure product was 0.19 g (61%) m.p. >260° C. (C₁₅ H₁₂ N₄ O₅,MW:328.29). Elemental Analysis [%]: Found C, 54.80; H, 3.59; N, 16.86.Calculated C, 54.88; H, 3.68; N, 17.07.

Group 6 compounds

F10

F10 can be prepared using a two step approach.

a) Preparation of 2-methyl-3-hydroxyethyl quinazolin-4-one

1.37 g (0.01 mol) anthranilic acid was refluxed with 8 ml aceticanhydride for 3 hours. The formed acetic acid was distilled offcontinuously at atmospheric pressure. After the acetic acid formationwas finished the mixture was evaporated in vacuo to dryness. Theresulting oil was mixed with 2 ml of ethanolamine and heated at 160° C.for 3 hours. After the reaction was completed the substance was cooled,mixed with alcohol and left at room temperature overnight. Theprecipitated crystals were collected by filtration. m.p. 159-60° C.;1.40 g (65%).

Step b

1.08 g (0.005 mol) 2-methyl-3 hydroxyethyl-quinazolin-4-one and 0.69 g3,4-dihydroxy-benzaldehyde were fused at 160° C. and heated foradditional 30 minutes. The resulting substance was dissolved inisopropanol, decolorized by charcoal and left at room temperatureovernight. The precipitated crystals were filtered and dried.

Yield of pure product was 0.79 g (49%) m.p. 221-223° C. (C₁₈ H₁₆ N₂ O₄,MW:324.34)

Elemental Analysis [%]: Found C, 66.48 H, 4.86; N, 8.62. Calculated C,66.66; H, 4.97; N, 8.64.

F11 and F12

1.01 g (5 mmol) of 3,4-dihydro-1,4-oxazine-[3,4-b]quinazolin-6-one werefused with 6 mmol of the corresponding benzaldehyde derivative on an oilbath at a temperature of 100-200° C. After removal of the water ofreaction, the resulting mixture was dissolved in ethanol and clarifiedwith charcoal. The solvent was evaporated and the productrecrystallized.

For preparation of F11, 3,4-dihydroxybenzaldehyde was used to obtain theproduct (85% yield) having a melting point of 290-292° C.

For preparation of F12 3-hydroxybenzaldehyde was used to obtain theproduct (63% yield) having a melting point of 208-214° C.

Group 7 Compounds

Benzoylhydroxyiminoethylacetate

To 10 g benzoyl ethylacetate in 20 ml acetic acid, cooled with ice, wasadded 3.7 g NaNO₂. After 10 minutes 5 ml water was added. After 3 hours100 ml water was added and the solid filtered to give 7.7 g, 84% yield,mp. 110° C.

NMR CDCl₃ δ7.90(2H,m), 7.6-7.5(3H,m), 4.30(2H,q,J=7.4 Hz),1.24(3H,t,J=7.4 Hz).

2-Ethoxycarbonyl-6,7-dimethyl-3-phenyl-quinoxaline

5 g, 22.6 mM benzoylhydroxyiminoethylacetate and 3.1, 22.8 mM, 4,5dimethyl 1,2-phenylene diamine in 20 ml ethanol and 5 ml HCl wererefluxed 6 hours. Workup H₂ O, NaHCO₃, CH₂ Cl₂), chromatography andtrituration with hexane gave 2 g, 29% yield, white solid, mp. 100° C.

NMR CDCl₃ d 7.96(1H,S), 7.93(1H,S), 7.7(2H,m), 7.5(3H,m) 4.30(2H,q,J=7.0Hz), 2.53(6H,S), 1.17(3H,t,J=7.0 Hz).

6,7-Dimethylquinoxalin-2-one

2 g, 15 mM, 4,5-dimethyl 1,2-diamino benzene and 1.5 g, 16 mM, glyoxalicacid hydrate in 30 ml ethanol were refluxed 2 hours.

Cooling and filtering gave 1.2 g, 46% yield, white solid mp-263° C., Sl.soluble acetone.

NMR DMSO d₆ δ60:40 mixture major--8.07 (1H,S), 7.55(1H,S), 7.06(1H,S),2.30(6H,S). minor--8.02 (1H,S), 7.42(1H,S), 7.28(1H,S), 2.28(6H,S).Remark--reaction of 4.1 g gave 3 g, 57%.

2-Chloro-6,7 Dimethyl Quinoxaline

1.1 g, 6.2 mM, 6,7-dimethylquinoxalin-2-one, 1 ml POCl₃ and 1 mldimethyl aniline in 20 ml toluene were refluxed 2 hours. Workup (NH₃,CH₂ Cl₂) and chromatography gave 0.4 g, 33% yield, white solid, mp-86°C.

NMR CDCl₃ δ8.68(1H,S,H₂), 7.85(1H,S), 7.76(1H,S), 2.50(6H,S).

G10

0.4 g (4 mM) phenylene diamine and 0.6 g (4 mM) phenyl glyoxalmonohydrate in 20 ml of ethanol, and 10 ml acetic acid was refluxed 3hours. Workup using 50 ml H₂ O and 80 ml CH₂ Cl₂ followed by triturationwith hexane gave 0.38 g (46% yield) of a white solid having a meltingpoint of 65° C.

NMR CDCl₃ δ9.44 (1H,S), 8.1(4H,m), 7.8(2H,m), 7.6(3H,m).MS-206(M+,100%), 179(M-HCN, 25), 152(37), 103(M-Ph-CN, 42), m/e.

G12

To 3 ml DMF and 16 ml PCCl₃ was added 2.7 g (10 mM)N-(3,4-dimethoxyphenyl)phenylacetamide. The reaction was heated at 90°C. for 4 hours, decanted on ice, filtered and washed with water to give2.9 g (96% yield) of a white solid having a melting point of 234° C.

NMR CDCl₃): δ8.26 (1H, s h₄), 8.0 (1H, s, H₈), 7.15 (5H, s Ph), 7.15(1H, s, h₅), 4.13, 4.05 (6H, 2s, OCH₃).

MS: 301, 299 (M+, 33%, 100%), 286, 284 (M-CH₃, 2%, 6%), 258, 256, (6%,18%), 220 (9%), 215, 213 (4%, 13%), m/e.

G11

The compound was synthesized by the procedure used for G12, except thatthe reactant N-(3,4,5-trimethoxy phenyl)phenylacetamide was substituted.The final product had a melting point of 103° C.

G13

2.4 g (16 mM) phenyl glyoxal hydrate and 2.2 g (16 mM)3,4-dimethyl-1,2-phenylene diamine in 20 ml ethanol were refluxed for1.5 hour. Cooling and filtering gave 3.25 g (88% yield) of a white solidhaving a melting point of 124° C.

NMR CDCl₃ δ9.23(1H,S,H₂), 8.19(1H,d,J=1.6 H₂), 8.15 (1H,d,J=1.7 H₂),7.90(2H,d,J=9.0 H₂), 7.57(3H,m)2.52(6H,S,CH₃).

MS-234(M+,100%), 219(M-CH₃, 11), 207(M-HCH, 12), 165(M-2HCN-CH₃,2),131(M-ph-CN,3), m/e.

G14

7 g of veratrole (51 mM) was added to 19 ml of ice-cooled 70% HNO₃.After 0.5 hour in the cold, 10 ml H₂ SO₄ was slowly added in 0.5 hour.The resulting dark suspension was stirred for 3 hours at roomtemperature and ice and water were added to the suspension toprecipitate the product. Filtering, washing with water and drying gave10.2 g (96% yield) of a yellow solid having a melting point of 120° C.(NMR CDCl₃ δ7.35(2H,S), 4.06(6H,S,OCH₃)). (Cf: J. Org. Chem. 12: 522(1947), reported m.p. 130° C., and J. Med. Chem. 36:331 (1993) reportedm.p. 122° C. The compound is sold by Lancaster Co., (reported m.p. 101°C.).

Two grams of 1,2-dinitro-4,5-dimethoxybenzene was hydrogenated over 0.3g PtO₂ for 1 hour, then filtered and evaporated to give 1.5 g of a blacksolid (Cf. J. Med. Chem. 36:331 (93), reported red brown solid, m.p.151° C.). The black solid was mixed with 1.3 g phenyl glyoxal, 15 mlabsolute ethanol and 15 ml of concentrated HCl and refluxed 5 hours.Workup, as for G10, gave a dark solid which was recrystallized fromethanol to give 0.72 g (31% yield) of a white solid having a meltingpoint of 134° C.

NMR CDCl₃ δ9.13 (1H,S,H₂), 8.16(1H,d,J=1d.6H₂), 7.60-7.40(5H,m),4.09(6H,S,OCH₃).

MS-266(M+,100%), 251(M-CH₃,12), 223(M-CH₃ --CO, 13), 196(M-CH₃--CO--HCN,5), m/e.

G15

Thiophene-2-glyoxal-bis-thiosemicarbazone (3.4 mM) and 0.6 g (4 mM)o-phenylenediamine in 15 ml acetic acid were refluxed 6 hours. Thesolvent was removed by distillation in vacuo, and the residue wasdissolved in CH₂ Cl₂ and washed with H₂ O. The organic layer wasevaporated in vacuo and the residue was triturated with benzene-hexaneto give a white solid (23% yield) mp 104° C.

NMR (CDCl₃): δ9.25(1H, s, H₂), 8.07, 7.72(4H, m, H₅₋₈), 7.85, 7.56,7.21(3H, m, thiophene).

MS: 212(M⁺, 100%), 185(M-HCN, 25%), 141(6%), 106(8%), m/e.

G16

2,3-diaminopyridine and phenyl glyoxal were reacted as for G13 to give awhite solid (77% yield) having a melting point of 135° C.

NMR CDCl₃ δ9.47(1H,S,H₂), 9.21 8.50, 7.71 (8 line ABC m, H₇,H₅,H₆),8.35(2H,m,Ph), 7.60(3H,m).

MS-207(M+,100), 180(H--HCN,8), 179(11), 104(23), 77(14),m/e.

CDCl₃ δ7.67(1H, dd), 6.89(dd), 6.62(dd), 4.25, 3.30 (br.S.).

G17

G17 was synthesized in two steps as follows:

a. Synthesis of 2-methoxy-4,5-dinitro phenol

3.3 g 1,2-dimethoxy-4,5-dinitrobenzene in 20 ml of 48% HBr was refluxedfor 16 hours. Water was added and the reaction was extracted with CH₂Cl₂ to give 1.1 g of an orange solid. Chromatography on silica gel,eluting with 2% CH₃ OH in CH₂ Cl₂ gave 0.42 g (13% yield) of a yellowsolid which turned red with KOH.

NMR (CDCl₃): δ7.44(1H,s), 7.42(1H,s), 6.30(1H,s), 4.07(3H,s).

Extraction of the aqueous phase with ethyl acetate gave 2 g of a redoil. Chromatography on silica gel, eluting with 5% CH₃ OH in CH₂ Cl₂gave a yellow solid, 0.1 g (3.5% yield) having a melting point of 160°C. with a KOH violet color, corresponding to 1,2dihydroxy-4,5-dinitrobenzene.

NMR (acetone-d₆) δ7.51(2H,s).

b. Reduction and condensation with phenylglyoxal

0.2 g 2-methoxy-4,5-dinitrophenol was hydrogenated on Pd/C in 20 mlethanol for one hour. The Pd was filtered, 0.3 g phenyl glyoxal wasadded, and the reaction was refluxed for three hours. Evaporation andchromatography on silica gel, eluting with 1% CH₃ OH in CH₂ Cl₂ gave a0.1 g of an orange oil.

NMR (CDCl₃): δ8.10, 7.6(7H,m), 3.54(3H,S).

G18

0.56 g (4 mM) 4,5-dimethyl 1,2-diaminobenzene and 0.6 g (4 mM) benzoylformic acid in 15 ml ethanol were refluxed 5 hours. Cooling andfiltering gave 0.8 g (80% yield) of a yellow solid having a meltingpoint of 275° C.

NMR (CDCl₃): δ8.38(2H, m), 7.51(3H, m), 7.70(1H, s), 7.06(1H, s)2.40(3H, s), 2.37(3H, s). Irradiation at 8.38 ppm gave a Singlet at 7.51ppm.

G19

3,4-diaminotoluene and phenyl glyoxal were reacted as for G13 to give alight brown solid (31% yield) having a melting point of 114° C.

NMR CDCl₃ δ9.29, 9.26(2S,2:1,H₂), 8.2, 8.17(2br.S), 8.07-7.90(3H,m),7.60(3H,m), 2.62(3H,S).

G20

0.15 g of G14 in 5 ml 48% HBr was refluxed 23 hours. Cooling andfiltering gave 95 mg (53% yield) of a green-yellow solid correspondingto the HBr salt of the quinazoline derivative, mp 280° C. HBr wasdetermined by elemental analysis.

NMR (DMSO-d₆): δ9.25(1H, s, H₂), 8.24(1H, d, J=1.9 Hz), 8.20(1H, d, J=1,9 Hz), 7.50(3H, m), 7.35(2H, m).

The mother liquid was neutralized with NaHCO₃. Extraction with EtAc gave20 mg (15% yield) of an orange solid, mp 305° C. corresponding to thefree base.

NMR (acetone d₆): δ9.19(1H s, H₂), 8.29(1H, d, J=1.5 Hz), 8.25(1H, d,J=1.5 Hz), 7.6(3H, m), 7.40(2H, m). MS: 238(M+, 54%), 211(M-HCN, 10%),154(7%), 108(1,2-benzoquinone, 100%), m/e.

G21

4-Benzoyl 1,2-phenylene diamine and phenyl glycoxal were reacted as forG13 to give a white solid (69% yield) having a melting point of 133° C.

NMR: CDCl₃ δ9.40(1H,S,H₂), 8.49(1H,S,H₅), 8.27(4H,br,S,H₇,8 H₂,'6'),7.90(2H,d,J=7.6 H₂), 7.60(6H,m).

G22

0.47 g (3 mM) 2,3-diaminoaphtalene and 0.47 g phenyl gloxal hydrate in20 ml ethanol were refluxed for 1.5 hour. Cooling and filtering gave 0.5g (65% yield) of a light brown solid having a melting point of 163° C.

NMR: CDCl₃ δ9.38(1H,S,H₂), 8.71,8.67(2H,2d,H₅,10), 8.25,8.10(4H,AA'BB'm.,H₆,9), 7.58(5H,m,Ph), MS-256(H⁺, 100%), 229(H--CN, 12%), 126(71),m/e.

G23

0.6 g, 4 mM, phenyl glyoxal and 0.6 g, 4 mM, 4-nitro phenylene diaminein 15 ml ethanol were refluxed 1.5 hours. Cooling and filtering gave 0.9g, 90% yield, white solid, mp-203° C.

NMR CDCl₃ δ9.49 (1H,S,H₂), 9.02(1H, d, J=2.5 Hz, H_(s)), 8.54(1H, dd,J=9.2, 2.5 Hz, H₇), 8.27(3H, m, Ph+H₇), 7.60 (3H, m, Ph).

G24

1.4 g, 10.3 mM, 4,5-dimethyl 1,2-phenylene diamine and 1.9 g, 10.2 mM,α-chloro 3,4-dihydroxy acetophone in 25 ml ethanol were refluxed 2hours. Cooling and filtering gave 0.76 g, 18% yield, deep yellow solid,mp. 278° C. as the HCl salt.

G25

2.4 g 2-ethoxycarbonyl-6,7-dimethyl-3-phenyl-quinoxaline and 5 g KOH in20 ml ethanol and 20 ml H₂ O were stirred 20 hours at room temperature.Acidification with HCl, filtering and washing with water gave 2.1 g, 96%yield, light-yellow solid, mp. 153° C.

NMR acetone d₆ δ7.92(1H,S), 7.90(1H,S), 7.85(2H,m), 7.50(3H,m),2.56(6H,S).

G27: 2-(4-nitrophenyl) 6,7 Dimethylquinoxalin

1. 4-nitrophenyl glyoxal

6.60 (40 mmol) 4-nitroacetophenone was dissolved in 30 ml of diozane and5 g, (45 mmol) selenium dioxyde was dissolved in 2.2 ml water and mixed.The mixture was refluxed for 16 hours with continuous stirring. Thereaction mixture was passed through an alumina column to removeselenium. The solvent was evaporated in vacuum. The crude product wasused in the next step without further purification.

R_(f) 0.80(EtOAc)

Y:7.00 (85%)

IR(cm⁻¹): 1730 (CO), 1520, 1330 (NO₂)

2., 2-(4-nitrophenyl) 6,7 dimethylquinoxaline

0.50. g (3 mmol) 4-nitrophenyl glyoxal was dissolved in 20 ml ethanol.0.34 g (2.5 mml) 1,2 dimethyl 4,5 diaminobenzene was dissolved inethanol. The reaction mixture was stirred and refluxed for 1 hour.Product crystallized after cooling, and filtrated washing with ethanolthen ether.

G28

G28 can be produced using the protocal described for G29 infra, exceptthat 3-bromoaniline is used instead of m-iodoaniline.

G29

150 mg, 0.8 mM, 2-chloro-6,7 dimethyl quinoxaline and 0.8 g, 3.6 mM,m-iodoaniline were heated at 100° C. for 3.5 hours. Chromatography gave100 mg, 35% yield, yellow solid, mp-185° C.

NMR CDCl₃ δ8.33(1H,S), 8.22(1H,m), 7.7(2H,m), 7.40(1H,m), 7.10(2H,m),2.45(3H,S), 2.43(3H,S).

G30

210 mg, 1.1 mM, 2-chloro-6,7 dimethyl quinoxaline and 0.8 g, 3.6 mM,p-iodoaniline were heated at 100° C. for 4 hours. Chromatography gave245 mg, 60% yield, light green solid, mp-228° C.

NMR, CDCl₃ δ8.32(1H,S), 7.67(1H,S), 7.64(H,S), 7.68, 7.56(4H,ABq,Jab=9.0 Hz).

Group 8

H10

0.01 mol (1.07 g) of benzylamine and 0.01 mol (1.38 g) of3,4-dihydroxybenzaldehyde were mixed together in 15 ml of ethanol andrefluxed on a waterbath for 15 minutes then 0.01 mol (3.44 g) of2-(1'-tosyloxyethyl)-quinazolin-4-one and one drop of pyridine wereadded and the mixture was refluxed for six hours. The resulting solutionwas evaporated and extracted with a 5% water solution of sodiumbicarbonate. The remaining crystals were filtered off, washed with waterand recrystallized from isopropanole.

Yield: 3.07 g (77%) M.p.:209-211° C. Formula: C₂₄ H₂₁ N₃ O₃

Elemental analysis [%] Calculated: C: 72.17 H: 5.30 N: 10.52 Found: C:72.12 H: 5.26 N: 10.46.

H11

0.01 mol (1.07 g) of benzylamine and 0.01 mol (1.22 g) ofsalicylaldehyde were mixed together in 15 ml of ethanol and refluxed ona waterbath for 15 minutes then 0.01 mol (3.44 g) of2-(1'-tosyloxyethyl)-quinazolin-4-one and one drop of pyridine wereadded and the mixture was refluxed for six hours. The resulting solutionwas evaporated and extracted with a 5% water solution of sodiumbicarbonate. The remaining crystals were filtered off, washed with waterand recrystallized from isopropanole.

Yield: 2.99 g (78%) M.p.:189-192° C. Formula: C₂₄ H₂₁ N₃ O₂

Elemental analysis [%] Calculated: C: 75.18 H: 5.52 N: 10.96 Found: C:75.09 H: 5.49 N: 10.90.

H12

0.01 mol (1.07 g) of benzylamine and 0.01 mol (1.22 g) of3-hydroxybenzaldehyde were mixed together in 15 ml of ethanol andrefluxed on a waterbath for 15 minutes then 0.01 mol (3.44 g) of2-(1'-tosyloxyethyl)-quinazolin-4-one and one drop of pyridine wereadded and the mixture was refluxed for six hours. The resulting solutionwas evaporated and extracted with a 5% water solution of sodiumbicarbonate. The remaining crystals were filtered off, washed with waterand recrystallized from isopropanole.

Yield: 2.72 g (71%) M.p.:184-185° C. Formula: C₂₄ H₂₁ N₃ O₂

Elemental analysis [%] Calculated: C: 75.18 H: 5.52 N: 10.96 Found: C:75.02 H: 5.45 N: 11.08.

H13

0.01 mol (1.07 g) of benzylamine and 0.01 mol (1.22 g) of4-hydroxybenzaldehyde were mixed together in 15 ml of ethanol andrefluxed on a waterbath for 15 minutes then 0.01 mol (3.44 g) of2-(1'-tosyloxyethyl)-quinazolin-4-one and one drop of pyridine wereadded and the mixture was refluxed for six hours. The resulting solutionwas evaporated and extracted with a 5% water solution of sodiumbicarbonate. The remaining crystals were filtered off, washed with waterand recrystallized from isopropanole.

Yield: 3.40 g (89%) M.p.:217-219° C. Formula: C₂₄ H₂₁ N₃ O₂

Elemental analysis [%] Calculated: C: 75.18 H: 5.52 N: 10.96 Found: C:75.26 H: 5.47 N: 10.88.

H14

0.01 mol (1.07 g) of benzylamine and 0.01 mol (1.54 g) of3,4,5-trihydroxybenzaldehyde were mixed together in 15 ml of ethanol andrefluxed on a waterbath for 15 minutes then 0.01 mol (3.44 g) of2-(1'-tosyloxyethyl)-quinazolin-4-one and one drop of pyridine wereadded and the mixture was refluxed for six hours. The resulting solutionwas evaporated and extracted with a 5% water solution of sodiumbicarbonate. The remaining crystals were filtered off, washed with waterand recrystallized from isopropanole.

Yield: 3.36 g (81%) M.p.:223-225° C. Formula: C₂₄ H₂₁ N₃ O₄

Elemental analysis [%] Calculated: C: 69.39 H: 5.10 N: 10.11 Found: C:69.51 H: 5.07 N: 10.08.

Group 9

I10

0.3 g (2 mM) 5-formyl indole and 0.4 g (2 mM), of2-cyano-H-(1-(+)phenylethyl)acetamide in 5 ml ethanol and 2 dropspiperidine were refluxed 3 hours. Water and HCl were added and thereaction extracted with ethyl acetate to give viscous oil.Chromatography on silica gel gave 0.42 g (66% yield) of pale-yellowsolid having a melting point of 76° C.

MS-315 (M+, 24%), 196 (M-NCH(CH₃)C₆ H₅, 22), 195 (25), 188 (21), 173(24), 168 (13), 149 (57), 145 (100), 134 (92), 116 (53), m/e.

Group 10

J10

230 mg, 1.06 mM, 5-bromo 3,4,dihydroxy benzaldehyde, 76 mg, 0.53 mM,diactonitrile sulphone and 10 mg β-alanine in 10 ml ethanol wererefluxed 5 hours. Cooling and filtering gave 220 mg, 76% yield, orangesolid, mp>300° C.

NMR acetone d₆ δ8.18(2H,S, vinyl), 7.90(2H, d,J=1.6 Hz), 7.78(2H,d,J=1.6Hz).

J11: 2-(3-bromo-4,5-dihydroxyphenyl)-1-cyano-1-cyanomethylsulfonlyethene

A mixture of 500 mg of 5-bromo-3,4-dihydroxybenzaldehyde and 700 mg ofsulfonly diacetonitrile in 6 ml of ethanol was refluxed with few dropsof piperidine for 4 hours. Ethanol was removed in rotavap and themixture worked up with ethyl acetate, diluted acid and brine. A portionof the crude was then purified by HPLC on a C-18 column to provide about50 mg of 2-(3-bromo-4,5-dihydroxyphenyl)-1-cyano-1-cyanomethylsulfonlyethene.

Group 11

6.7-Dimethoxy quinazolin-4-one

7 g 4,5-dimethoxy 2-amino benzoic acid and 8 ml formamide were heated 2hours at 170° C. Cold water was added and the solid filtered to give 0.9g, 12% yield, light-brown solid, mp. 308° C.

NMR--DMSO d₆ δ8.0 (1H, S) 7.43(1H,S), 7.12(1H,S) 3.89(3H,S), 3.85(3H,S).

4-Chloro-6,7, Dimethoxy quinalozine

0.8 g, 6,7-dimethoxy quinazolin-4-one, 1 ml POCl₃ and 1 ml dimethylaniline in 20 ml toluene were refluxed 3.5 hours. Workup and triturationwith hexane gave 0.5 g light grey solid, 0.5 g, 57% yield, mp. 188° C.

NMR CDCl₃ δ8.88 (1H,S), 7.41(1H,S), 7.36(1H,S), 4.09 (3H,S), 4.08(3H,S).

P10

0.3 g, 1.4 mM, 3,4-dihydroxy 5-bromo benzaldehyde, 0.15 g, 0.7 mM,N-3-cyanomethylcarbonylamino-N-propylcyanoacetamide, and 25 mg β-alaninein 20 ml ethanol were refluxed 3 hours. Cooling and filtering gave 0.24g, 57% yield, yellow solid, mp 283° C.

P12: trophene-2-carboxylic acid (3,5-bis-(trifluoromethyl) anilide

0.45 ml (2.9 mmol) of 3,5-bis-(trifluoromethyl) aniline was dissolved in5 ml of abs pyridine, then cooled to 10° C. 0.12 ml (1.5 mmol)phosphorous trichloride was added with continuous stirring dropwise.After 0.5 hour 0.51 g (4 mmol) thiophene-2-carboxylic acid was added,and stirred for 12 hours at room temperature. Solvent was evaporated invacuum, 1N HCl was added to the residue and extracted with ethylacetate.The ethylacetate solution was extracted with bicarbonate solution, driedon sodium sulfate, filtrated and evaporated.

Product was triturated with ether, filtrated and dried in vacuum.

m.p.: 145-147° C.

Rf: 0.80 (Hexane-Et)Ac=1:1)

y:0.62 g (80%)

IR(cm⁻¹): 3280 (N-H): 1640 (CONH); 1560 (Car); 1130 (C-F)

P13

3 g, 20.1 mM, 3-methyl isoquinoline in 20 ml acetic acid and 5 ml 30% H₂O₂ was heated at 70° C. for 14 hours. Water was added to the cooledsolution and bicarbonate to neutrality. Extraction with CH₂ Cl₂ andtrituration with hexane gave 1.9 g, 57% yield, white solid, mp 128° C.(J.O.C., 21:1337(1956), mp-138° C.).

NMR CDCl₃ δ8.86(1H,S), 7.70-7.50(5H,m), 2.64 (3H,S).

P14

a. 0.04 g, 1.8 mM, 4-chloro-6,7, dimethoxy quinalozine and 0.19 g, 2 mM,aniline in 15 ml ethanol were refluxed for 1 hour. Cooling and filteringgave 0.445 g, 78% yield, light yellow solid, mp-268° C., as the HCl saltP14a.

b. free base--0.35 g P14a was treated with H₂ O-Na₂ CO₃ and extractedwith CH₂ Cl₂ to give 0.13 g, 42% yield, white solid, mp-241° C.

NMR CDCl₃ δ58.66 (1H,S,H₂), 7.67(1H,S), 7.63(1H,S), 7.4-7.14(5H,m),3.96(3H,S), 3.93(3H,S).

P15:1-(2-chlorophenylmethylene)-3-(3-methoxy-n-propyl)-2,4-thiazolidinedione

A solution of 400 mg of 3-(3-methoxy-n-propyl)-2,4-thiazolidinedione and260 mg of 2-chlorobezaldehyde in 4 ml of ethanol with one drop ofpiperidine was refluxed for 4 hours. The mixture was then worked up withethyl acetate and water. The crude product was purified on a silica gelcolumn (5% methanol in dichloromethane) to provide 200 mg of1-(2-chlorophenylmethylene)-3-(3-methoxy-n-propyl)-2,4-thiazolidinedione.(P15 can also be obtained from Aldrich Chemical.).

P16

0.69 g, 2.5 mM, 5-iodo vaniline, N-3-phenyl-N-propyl cyanoacetamide(prepared as described by Gazit et al., J. Med Chem 34:1896, 1991) and50 mg β-alanine in 30 ml ethanol were refluxed 5 hours. Evaporation gavean oil which was triturated with benzene-hexane and filtered to give abright yellow solid, 0.82g, 71% yield, mp. 83° C.

NMR CDCl₃ δ8.12(1H,S), 7.75(1H,d,J=2.0 Hz), 7.68(1H,d,J=2.0 Hz),7.30-7.10(5H,m), 3.96(3H,S,O,CH₃), 3.45(2H,q,J=6.0 Hz), 2.70(2H,t,J=6.0Hz), 1.95(2H, quin, J=6.0 Hz).

MS-462(M⁺,53), 357(M-CH₂)₃ Ph,18), 335(M-I,100), 327(M-NH(CH₂)₃ ph, 31),m/e

P17

a. 0.4 g, 1.8 mM, 4-chloro-6,7, dimethoxy quinalozine and 0.24 g, 2 mM,indoline in 10 ml ethanol were refluxed 2 hours, cooled and filtered togive 0.46 g, 74% yield, yellow solid (P17a), mp. 238° C.

b. free base--0.3 g AG P17a was treated with H₂ O-Na₂ CO₃ and extractedwith CH₂ Cl₂ to give 0.13 g, 48% yield, white solid, mp. 158° C.

NMR CDCl₃ δ8.79(1H,S,H₂), 7.30(1H,S), 7.28(1H,S), 7.14-6.80(4H,m),4.36(2H,t,J=7.6 Hz), 4.06(3H,S,OCH₃), 3.85(3H,S,OCH₃), 3.22(2H,t,J=7.6Hz).

P18: 1-cyano-2-(3-ethoxy-4-hydroxyohenyl)-1-methoxycarbonyl ethene

A mixture of 20 grams of 3-ethoxyl-4-hydroxybenzaldhyde and 13 grams ofmethyl cyanoacetate in 100 ml of ethanol was refluxed with 1 ml ofpiperidine for 4 hours. The crude mixture was allowed to cool down toroom temperature and, with stirring, water was added until the solidbegan to form. This mixture was then refrigerated for 4 hours and solidswas collected by suction filtration, washed with a cold mixture ofethanol and water (1:2) and suction dried to provide 25 grams of1-cyano-2-(3-ethoxy-4-hydroxyphenyl)-1-methoxycarbonyl ethene.

P19: N-2-chlorothenyl (2-cyano-2-N-moriholinylcarbonyl) thioacetamide

A solution of 1.5 gram of N-morpholinyl cyanoacetamide in 20 ml oftetrohydrofuran at 0° C. was added with 680 mg of sodium ethoxide. Thismixture was stirred at 0° C. for 1 hour and added with 1.7 grams of2-chlorophenylisothiocyanate in 5 ml of tetrahydrafuran dropwise. Afteraddition, the mixture was warmed up to room temperature and heated at50° C. for 6 hours. Upon cooling, all the ethanol was removed and theresulting solid suspended in 10 ml of water. This was then added with 3ml of 1N sodium hydroxide solution, shaken vigorously and washed with 50ml of ethyl ether. The aqeous layer was then acidified with 1Nhydrochoric acid until pH 1. The solid was then collected by suctionfiltration. This produced 750 mg of N-2-chlorophenyl(2-cyano-2-N-morpholinylcarbonyl) thioacetamide. (P19 can also beobtained from Ryan Scientific.)

P20: N-2-chlorophenyl(2-cyano-2-N-3-trifluorophenylaminocarbonyl)thioacetamide

P20 was synthesized using similar conditions as decribed for P19 butstarting with N-3-trifluroromethylphenyl cyanoacetamide. (P20 can alsobe obtained from Ryan Scientific.)

P21: N-3-methoxyphenyl(2-cyano-2-N-pyrrolidinycarbonyl)thioacetamide

P21 was synthesized using similar conditions as described for P19 butstarting with N-pyrrolidinly cyanoacetamide and3-methoxylphenylisothiocyanate as the reagents. (P19 can also beobtained from Ryan Scientific.)

P22: N-4-trifluoromethlybenzyl 3,5-dimethylisoxazole-4-carboxamide

P22 was made using the same conditions as described forN-trifluoromethlyphenyl 3,5-dimethylisoxazole-4-carboxamride (A13), butstarting with 4-trifluoromethylbenzylamine.

P23: N-trifluoromethlybenzyl 3-methylisoxazole-4-carboxamide

A solution of 3 grams of 3-methlyisoxazole-4-carboxylic acid and 4.8grams of 1,3-dicyclohexylcarbodimide in 30 ml of dichloromethane wasstirred at room temperature for 30 minutes. This was then added with 8ml of 4-trifluoromethylbenzylamine dropwise and the mixture stirred atroom temperature overnight. The mixture was then diluted in ethylacetate (100 ml) and worked up with diluted hydrochloride solution,saturated sodium bicarbonate and sodium chloride solution, dried oversodium sulfate, filtered and concentrated. The crude was thencrystallized with ethanol and water to provide 1.5 grams ofN-trifluoromethlybenzyl 3-methylisoxazole-4-carboxamide.

P24: 1-trifluoromethlybenzylaminocarbonyl-1-cyano-2-hydroxy pronene

A solution of 500 mg of N-trifluoromethlybenzyl3-methylisoxazole-4-carboxamide in 5 ml of ethanol was added to 300 mgof 1,8-diazabicyclo[5.4.0]undec-7-ene. The mixture was then stirred atroom temperature for 1 hour and acidified with 2 ml of 2N hydrochloridesolution. The solid was collected by filtration to provide 350 mg of1-trifluoromethlybenzylaminocarbonyl-1-cyano-2-hydroxy propene

P25

0.42 g, 3 mM, 3,4-dihydroxy benzaldehyde, 0.6 g, 3.1 mM,N-4-flurobenzylcyanoacetamide and 2.0 mg β-alanine were refluxed 5hours. Concentration and filtration gave 0.89 g, 95% yield, yellowsolid, mp -212° C., NMR acetone d₆ δ8.10 (1H,S, Vinyl), 7.68(1H,d,J=2.3Hz, H₂) 7.40(3H,m,H₆,H₂,H₆), 7.09(2H,t,J=8.8 Hz, H_(3'),5')6.98(1H,d,J=8.3 Hz,H₅), 4.56(2H,br,S).

MS-312(M⁺,46%), 311(30), 295(H-OH,35), 161(17), 124(NHCH₄ C₆ H₄ F,100%), 109 (CH₂ C₆ H₄ F,73)-m/e.

Other embodiments are within the following claims.

We claim:
 1. A method of treating a patient suffering from a bloodvessel proliferative disorder characterized by inappropriate PDGF-Ractivity, comprising the step of administering to said patient atherapeutically effective amount of a compound selected from the groupconsisting of 5-methyl-isoxazole-4-carboxylicacid-(4-trifluoromethyl)-anilide,2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)-2-butenamide, andpharmaceutically acceptable salts thereof,wherein said compoundsignificantly inhibits one or more PDGF-R activities in vivo or invitro.
 2. The method of claim 1, wherein said compound is5-methyl-isoxazole-4-carboxylic acid-(4-trifluoromethyl)-anilide, or apharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein said compound is2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)-2-butenamide, or apharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein said compound selectively inhibits PDGF-R activity.
 5. Themethod of claim 4, wherein said compound selectively inhibits one ormore members of related tyrosine kinases of the group consisting of KDR,Flt-1 and Flk-1.
 6. The method of claim 1, wherein said patient is ahuman.
 7. The method of claim 1, wherein said blood vessel proliferativedisorder is selected from the group consisting of restenosis,retinopathies, and atherosclerosis.
 8. The method of claim 7, whereinsaid blood vessel proliferative disorder is restenosis.
 9. The method ofclaim 7, wherein said blood vessel proliferative disorder is aretinopathy.
 10. The method of claim 7, wherein said blood vesselproliferative disorder is atherosclerosis.
 11. A method of treating apatient suffering from a blood vessel proliferative disordercharacterized by inappropriate PDGF-R activity, comprising the step ofadministering to said patient a therapeutically effective amount of acomposition comprising a compound selected from the group consisting of5-methyl-isoxazole-4-carboxylic acid-(4-trifluoromethyl)-anilide,2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)-2-butenamide, andpharmaceutically acceptable salts thereof,wherein said compoundsignificantly inhibits one or more PDGF-R activities in vivo or invitro.
 12. The method of claim 11, wherein said compound is5-methyl-isoxazole-4-carboxylic acid-(4-trifluoromethyl)-anilide, or apharmaceutically acceptable salt thereof.
 13. The method of claim 11,wherein said compound is2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)-2-butenamide, or apharmaceutically acceptable salt thereof.
 14. The method of claim 11,wherein said compound selectively inhibits PDGF-R activity.
 15. Themethod of claim 11, wherein said compound selectively inhibits one ormore members of related tyrosine kinases of the group consisting of KDR,Flt-1 and Flk-1.
 16. The method of claim 11, wherein said patient is ahuman.
 17. The method of claim 11, wherein said blood vesselproliferative disorder is selected from the group consisting ofrestenosis, retinopathies, and atherosclerosis.
 18. The method of claim17, wherein said blood vessel proliferative disorder is restenosis. 19.The method of claim 17, wherein said blood vessel proliferative disorderis a retinopathy.
 20. The method of claim 17, wherein said blood vesselproliferative disorder is atherosclerosis.